Fusion proteins of mycobacterium tuberculosis

ABSTRACT

The present invention relates to compositions and fusion proteins containing at least two  Mycobacterium  sp. antigens, and nucleic acids encoding such compositions and fusion proteins. The compositions of the invention increase serological sensitivity of sera from individuals infected with tuberculosis, and methods for their use in the diagnosis, treatment, and prevention of tuberculosis infection.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application claims priority to U.S. patent application Ser.No. 09/597,796, filed Jun. 20, 2000, and U.S. patent application No.60/265,737, filed Feb. 1, 2001, herein each incorporated by reference intheir entirety.

The present application is related to U.S. patent application Ser. No.09/056,556, filed Apr. 7, 1998; U.S. patent application Ser. No.09/223,040, filed Dec. 30, 1998; U.S. patent application Ser. No.09/287,849, filed Apr. 7, 1999; published PCT application No.WO99/51748, filed Apr. 7, 1999 (PCT/US99/07717), U.S. patent applicationNo. 60/158,338, filed Oct. 7, 1999, and U.S. application No. 60/158,425,filed Oct. 7, 1999; U.S. application Ser. No. 09/688,672, filed Oct. 10,2000; and published PCT application No. WO01/24820, filed Oct. 10, 2000(PCT/US00/28095); herein each incorporated by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Not applicable.

FIELD OF THE INVENTION

The present invention relates to fusion proteins containing at least twoMycobacterium sp. antigens. In particular, it relates to nucleic acidsencoding fusion proteins that include two or more individual M.tuberculosis antigens, which increase serological sensitivity of serafrom individuals infected with tuberculosis, and methods for their usein the diagnosis, treatment, and prevention of tuberculosis infection.

BACKGROUND OF THE INVENTION

Tuberculosis is a chronic infectious disease caused by infection with M.tuberculosis and other Mycobacterium species. It is a major disease indeveloping countries, as well as an increasing problem in developedareas of the world, with about 8 million new cases and 3 million deathseach year. Although the infection may be asymptomatic for a considerableperiod of time, the disease is most commonly manifested as an acuteinflammation of the lungs, resulting in fever and a nonproductive cough.If untreated, serious complications and death typically result.

Although tuberculosis can generally be controlled using extendedantibiotic therapy, such treatment is not sufficient to prevent thespread of the disease. Infected individuals may be asymptomatic, butcontagious, for some time. In addition, although compliance with thetreatment regimen is critical, patient behavior is difficult to monitor.Some patients do not complete the course of treatment, which can lead toineffective treatment and the development of drug resistance.

In order to control the spread of tuberculosis, effective vaccinationand accurate early diagnosis of the disease are of utmost importance.Currently, vaccination with live bacteria is the most efficient methodfor inducing protective immunity. The most common mycobacterium employedfor this purpose is Bacillus Calmette-Guerin (BCG), an avirulent strainof M. bovis. However, the safety and efficacy of BCG is a source ofcontroversy and some countries, such as the United States, do notvaccinate the general public with this agent.

Diagnosis of tuberculosis is commonly achieved using a skin test, whichinvolves intradermal exposure to tuberculin PPD (protein-purifiedderivative). Antigen-specific T cell responses result in measurableinduration at the injection site by 48-72 hours after injection, whichindicates exposure to mycobacterial antigens. Sensitivity andspecificity have, however, been a problem with this test, andindividuals vaccinated with BCG cannot be distinguished from infectedindividuals.

While macrophages have been shown to act as the principal effectors ofMycobacterium immunity, T cells are the predominant inducers of suchimmunity. The essential role of T cells in protection againstMycobacterium infection is illustrated by the frequent occurrence ofMycobacterium infection in AIDS patients, due to the depletion of CD4⁺ Tcells associated with human immunodeficiency virus (HIV) infection.Mycobacterium-reactive CD4⁺ T cells have been shown to be potentproducers of γ-interferon (IFN-γ), which, in turn, has been shown totrigger the anti-mycobacterial effects of macrophages in mice. While therole of IFN-γ in humans is less clear, studies have shown that1,25-dihydroxy-vitamin D3, either alone or in combination with IFN-γ ortumor necrosis factor-alpha, activates human macrophages to inhibit M.tuberculosis infection. Furthermore, it is known that IFN-γ stimulateshuman macrophages to make 1,25-dihydroxy-vitamin D3. Similarly,interleukin-12 (IL-12) has been shown to play a role in stimulatingresistance to M. tuberculosis infection. For a review of the immunologyof M. tuberculosis infection, see Chan & Kaufmann, Tuberculosis:Pathogenesis, Protection and Control (Bloom ed., 1994), and Harrison'sPrinciples of Internal Medicine, volume 1, pp. 1004-1014 and 1019-1023(14^(th) ed., Fauci et al., eds., 1998).

Accordingly, there is a need for improved diagnostic reagents, andimproved methods for diagnosis, preventing and treating tuberculosis.

SUMMARY OF THE INVENTION

The present invention therefore provides compositions comprising atleast two heterologous antigens, fusion proteins comprising theantigens, and nucleic acids encoding the antigens, where the antigensare from a Mycobacterium species from the tuberculosis complex and otherMycobacterium species that cause opportunistic infections in immunecompromised patients. The present invention also relates methods ofusing the polypeptides and polynucleotides in the diagnosis, treatmentand prevention of Mycobacterium infection.

In one aspect, the present invention provides compositions and fusionproteins comprising a mutated version of Ra35 (N-terminal portion ofMTB32A) or Ra35FL (full length MTB32A), in which one, two, or three ofthe three amino acids histidine, aspartate, or serine at the active sitehas been mutated to a different amino acid. In one embodiment, inRa35FL, the serine at position 183 has been mutated to an alanineresidue, creating Ra35FLMutSA. In one embodiment, the DNA encodingRa35FL has been mutated by changing a T to a G, resulting in a serine toalanine mutation at amino acid 183 of SEQ ID NO:4. In anotherembodiment, the present invention provides the fusion proteinMTB72FMutSA, in which the Ra35 component of the fusion protein has aserine to alanine mutation at amino acid position 710 of the MTB72Fsequence. In another embodiment, the present invention provides anucleic acid encoding the fusion protein MTB72F, in which the nucleicacid encoding the Ra35 component has been mutated by changing a T to aG, resulting in a serine to alanine mutation at amino acid position 710of the MTB72F sequence.

The present invention is based, in part, on the inventors' discoverythat fusion polynucleotides, fusion polypeptides, or compositions thatcontain at least two heterologous M. tuberculosis coding sequences orantigens are highly antigenic and upon administration to a patientincrease the sensitivity of tuberculosis sera. In addition, thecompositions, fusion polypeptides and polynucleotides are useful asdiagnostic tools in patients that may have been infected withMycobacterium.

In one aspect, the compositions, fusion polypeptides, and nucleic acidsof the invention are used in in vitro and in vivo assays for detectinghumoral antibodies or cell-mediated immunity against M. tuberculosis fordiagnosis of infection or monitoring of disease progression. Forexample, the polypeptides may be used as an in vivo diagnostic agent inthe form of an intradermal-skin test. The polypeptides may also be usedin in vitro tests such as an ELISA with patient serum. Alternatively,the nucleic acids, the compositions, and the fusion polypeptides may beused to raise anti-M. tuberculosis antibodies in a non-human animal. Theantibodies can be used to detect the target antigens in vivo and invitro.

In another aspect, the compositions, fusion polypeptides and nucleicacids may be used as immunogens to generate or elicit a protectiveimmune response in a patient. The isolated or purified polynucleotidesare used to produce recombinant fusion polypeptide antigens in vitro,which are then administered as a vaccine. Alternatively, thepolynucleotides may be administered directly into a subject as DNAvaccines to cause antigen expression in the subject, and the subsequentinduction of an anti-M. tuberculosis immune response. Thus, the isolatedor purified M. tuberculosis polypeptides and nucleic acids of theinvention may be formulated as pharmaceutical compositions foradministration into a subject in the prevention and/or treatment of M.tuberculosis infection. The immunogenicity of the fusion protein orantigens may be enhanced by the inclusion of an adjuvant, as well asadditional fusion polypeptides, from Mycobacterium or other organisms,such as bacterial, viral, mammalian polypeptides. Additionalpolypeptides may also be included in the compositions, either linked orunlinked to the fusion polypeptide or compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows percent survival of Guinea pigs vaccinated with MTB72Fpolyprotein.

FIG. 2 shows CFUs from spleen cells (FIG. 2A) and lung cells afterimmunization with MTB72F, MTB59F, MTB72F DNA, or a compositioncomprising Ra12, TbH9, and Ra35 antigens.

FIG. 3 shows a schematic diagram of MTB72F.

FIG. 4 shows the nucleotide and amino acid sequence of Ra35 (195 aminoacids from the N-terminal portion of MTB32A).

FIG. 5 shows an alignment of the amino acid sequences of MTB72F and themutated version MTB72FMutSA.

FIG. 6 shows an alignment of the amino acid sequences of mature (fulllength) Ra35/MTB32A and the mutated version Ra35FLMutSA.

FIG. 7 shows long term survival of guinea pigs vaccinated with Mtb72Fformulations.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention relates to compositions comprising antigencompositions and fusion polypeptides useful for the diagnosis andtreatment of Mycobacterium infection, polynucleotides encoding suchantigens, and methods for their use. The antigens of the presentinvention are polypeptides or fusion polypeptides of Mycobacteriumantigens and immunogenic thereof. More specifically, the compositions ofthe present invention comprise at least two heterologous polypeptides ofa Mycobacterium species of the tuberculosis complex, e.g., a speciessuch as M. tuberculosis, M. bovis, or M. africanum, or a Mycobacteriumspecies that is environmental or opportunistic and that causesopportunistic infections such as lung infections in immune compromisedhosts (e.g., patients with AIDS), e.g., BCG, M. avium, M.intracellulare, M. celatum, M. genavense, M. haemophilum, M. kansasii,M. simiae, M. vaccae, M. fortuitum, and M. scrofulaceum (see, e.g.,Harrison's Principles of Internal Medicine, volume 1, pp. 1004-1014 and1019-1023 (14^(th) ed., Fauci et al., eds., 1998). The inventors of thepresent application surprisingly discovered that compositions and fusionproteins comprising at least two heterologous Mycobacterium antigens, orimmunogenic fragments thereof, where highly antigenic. Thesecompositions, fusion polypeptides, and the nucleic acids that encodethem are therefore useful for eliciting protective response in patients,and for diagnostic applications.

The antigens of the present invention may further comprise othercomponents designed to enhance the antigenicity of the antigens or toimprove these antigens in other aspects, for example, the isolation ofthese antigens through addition of a stretch of histidine residues atone end of the antigen. The compositions, fusion polypeptides, andnucleic acids of the invention can comprise additional copies ofantigens, or additional heterologous polypeptides from Mycobacteriumsp., such as MTB8.4 antigen, MTB9.8 antigen, MTB9.9 antigen, MTB40antigen, MTB41 antigen, 38-1, ThRa3, 38 kD, DPEP, TbH4, DPPD, ESAT-6antigen, MTB85 complex antigen (e.g., MTB85b), or α-crystalline antigen,and Erd14. The compositions, fusion polypeptides, and nucleic acids ofthe invention can also comprise additional heterologous polypeptidesfrom other non-Mycobacterium sources. For example, the compositions andfusion proteins of the invention can include polypeptides or nucleicacids encoding polypeptides, wherein the polypeptide enhances expressionof the antigen, e.g., NS1, an influenza virus protein, or an immunogenicportion thereof (see, e.g. WO99/40188 and WO93/04175). The nucleic acidsof the invention can be engineered based on codon preference in aspecies of choice, e.g., humans.

The compositions of the invention can be naked DNA, or the compositions,e.g., polypeptides can also comprise adjuvants, e.g., MPL, 3D-MPL, IFA,AS adjuvants such as AS2, AS2′, AS2″, AS4, AS6, ENHANZYN (Detox), QS21,CWS, TDM, AGP, CPG, Leif, saponin, and saponin mimetics, and derivativesthereof. In addition, the compositions of the invention can comprise BCGor Pvac as an adjuvant.

In one embodiment, the compositions and fusion proteins of the inventionare composed of at least two antigens selected from the group consistingof a MTB39 antigen or an immunogenic fragment thereof from aMycobacterium species of the tuberculosis complex, and a MTB32A antigenor an immunogenic fragment thereof from a Mycobacterium species of thetuberculosis complex.

In another embodiment, the antigens are selected from the groupconsisting of a MTB39 antigen or an immunogenic fragment thereof from aMycobacterium species of the tuberculosis complex, and a polypeptidecomprising at least 205 amino acids of the N-terminus of a MTB32Aantigen from a Mycobacterium species of the tuberculosis complex.

In another embodiment, the antigens are selected from the groupconsisting of a MTB39 antigen or an immunogenic fragment thereof from aMycobacterium species of the tuberculosis complex, a polypeptidecomprising at least about 205 amino acids of the N-terminus of a MTB32Aantigen from a Mycobacterium species of the tuberculosis complex, and apolypeptide comprising at least about 132 amino acids from theC-terminus of MTB32A antigen from a Mycobacterium species of thetuberculosis complex.

In the nomenclature of the application, Ra35 refers to the N-terminus ofMTB32A (Ra35FL), comprising at least about 195 to 205 amino acids ofMTB32A from M. tuberculosis, or the corresponding region from anotherMycobacterium species. Ra12 refers to the C-terminus of MTB32A (Ra35FL),comprising at least about the last 132 amino acids from MTB32A from M.tuberculosis, or the corresponding region from another Mycobacteriumspecies.

The following provides sequences of some antigens-used in thecompositions and fusion proteins of the invention:

SEQ ID NO: 1-4: MTB32A (Ra35FL or Ra35 mature), the sequence of which isalso disclosed as SEQ ID NO:17 (cDNA) and SEQ ID NO:79 (protein) in theU.S. patent application Ser. Nos. 08/523,436, 08/523,435, 08/658,800,08/659,683, 08/818,112, 09/056,556, and 08/818,111 and in the WO97/09428and WO97/09429 applications, see also Skeiky et al., Infection andImmunity 67:3998-4007 (1999). The term MTB32A also includes MTB32A aminoacid sequences in which any one of the three amino acids at the activesite triad (His, Asp, Ser), e.g., the serine residue at amino acidposition 207 in SEQ ID NO:2 or amino acid position 183 in SEQ ID NO:4,has been changed to another amino acid (e.g., alanine, Ra35FLMutSA, see,e.g., FIG. 6 and SEQ ID NO:6).

SEQ ID NO:5 and 6: Ra35FLMut SA, the mature version of RA35FL in whichthe serine residue at amino acid position 183 of SEQ ID NO:4 has beenchanged to an alanine residue.

SEQ ID NO:7 and 8: Ra35, the N-terminus of MTB32A (Ra35FL), comprisingat least about 195 amino acids from the N-terminus of MTB32A from M.tuberculosis, the nucleotide and amino acid sequence of which isdisclosed in FIG. 4 (see also amino acids 33-227 of SEQ ID NO:2 andamino acids 8 to 202 of SEQ ID NO:4). The term Ra35 (N-term) alsoincludes Ra35 amino acid sequences in which any one of the three aminoacids at the active site triad (i.e., His, Asp, or Ser) has been changedas described above.

SEQ ID NO:9 and 10: MTBRa12, the C-terminus of MTB32A (Ra35FL),comprising at least about 132 amino acids from the C-terminus of MTB32Afrom M. tuberculosis (see, e.g., amino acids 224 to 355 of SEQ ID NO:2and amino acids 199 to 330 of SEQ ID NO:4), the sequence of which isdisclosed as SEQ ID NO:4 (DNA) and SEQ ID NO:66 (predicted amino acidsequence) in the U.S. patent application Ser. No. 09/072,967.

SEQ ID NO:11, 12, 13, and 14: MTB39 (TbH9), the sequence of which isdisclosed as SEQ ID NO:106 (cDNA full length) and SEQ ID NO:107 (proteinfull length) in the U.S. patent application Ser. Nos. 08/658,800,08/659,683, 08/818,112, and U.S. patent application Ser. No. 08/818,111and in the WO97/09428 and WO97/09429 applications. The sequence is alsodisclosed as SEQ ID NO:33 (DNA) and SEQ ID NO:91 (amino acid) in U.S.patent application Ser. No. 09/056,559.

The following provides sequences of some fusion proteins of theinvention

SEQ ID NO:15 and 16: MTB72F (Ra12-TbH9-Ra35), the sequence of which isdisclosed as SEQ ID NO:1 (DNA) and SEQ ID NO:2 (protein) in the U.S.patent application Ser. No. 09/223,040, U.S. patent application Ser. No.09/223,040, and in the PCT/US99/07717 application. The term MTB372F alsoincludes MTB72F amino acid sequences in which any one of the three aminoacids at the active site triad in Ra35FL (i.e., His, Asp, or Ser), hasbeen changed as described above (see, e.g., MTB72FMutSA, FIG. 5).

SEQ ID NO:17 and 18: MTB72FMutSA (Ra12-TbH9-Ra35MutSA), wherein, in theRa35 component of the fusion protein, the serine at position 710 hasbeen changed to an alanine.

SEQ ID NO:19 and 20: TbH9—Ra35 (MTB59F), the sequence of which isdisclosed as SEQ ID NO:23 (cDNA) and SEQ ID NO:24 (protein) in the U.S.patent application Ser. No. 09/287,849 and in the PCT/US99/07717application.

The following provides sequences of some additional antigens used in thecompositions and fusion proteins of the invention:

SEQ ID NO: 21 and 22: MTB8.4 (DPV), the sequence of which is disclosedas SEQ ID NO:101 (cDNA) and SEQ ID NO:102 (protein) in the U.S. patentapplication Ser. Nos. 08/658,800, 08/659,683, 08/818,112 and 08/818,111and in the WO97/09428 and WO97/09429 applications.

SEQ ID NO:23 and 24: MTB9.8 (MSL), the sequence of which is disclosed asSEQ ID NO:12 (DNA), SEQ ID NO:109 (predicted amino acid sequence) andSEQ ID NO:110 to 124 (peptides) in the U.S. patent application Ser. Nos.08/859,381, 08/858,998, 09/073,009 and 09/073,010 and in thePCT/US98/10407 and PCT/US98/10514 applications.

SEQ ID NO:25, 26, and 27: MTB9.9A (MTI, also known as MTI-A), thesequence of which is disclosed as SEQ ID NO:3 and SEQ ID NO:4 (DNA) andSEQ ID NO:29 and SEQ ID NO:51 to 66 (ORF peptide for MTI) in the U.S.patent application Ser. Nos. 08/859,381, 08/858,998, 09/073,009 andv09/073,010 and in the PCT/US98/10407 and PCT/US98/10514 applications.Two other MTI variants also exist, called MTI-B and MTI-C.

SEQ ID NO:28 and 29: MTB40 (HTCC#1), the sequence of which is disclosedas SEQ ID NO:137 (cDNA) and 138 (predicted amino acid sequence) in theU.S. patent application Ser. Nos. 09/073,009 and 09/073,010 and in thePCT/US98/10407 and PCT/US98/10514 applications.

SEQ ID NO:30 and 31: MTB41 (MTCC#2), the sequence of which is disclosedas SEQ ID NO:140 (cDNA) and SEQ ID NO:142 (predicted amino acidsequence) in the U.S. patent application Ser. Nos. 09/073,009 and09/073,010 and in the PCT/US98/10407 and PCT/US98/10514 applications.

SEQ ID NO:32 and 33: ESAT-6, the sequence of which is disclosed as SEQID NO:103 (DNA) and SEQ ID NO:104 (predicted amino acid sequence) in theU.S. patent application Ser. No. 09/072,967. The sequence of ESAT-6 isalso disclosed in U.S. Pat. No. 5,955,077.

SEQ ID NO:34 and 35: Tb38-1 or 38-1 (MTb11), the sequence of which isdisclosed in SEQ ID NO:46 (DNA) and SEQ ID NO:88 (predicted amino acid)in the U.S. patent application Ser. Nos. 09/072,96; 08/523,436;08/523,435; 08/818,112; and 08/818,111; and in the WO97/09428 andWO97/09429 applications.

SEQ ID NO:36 and 37: ThRa3, the sequence of which is disclosed in SEQ IDNO:15 (DNA) and SEQ ID NO:77 (predicted amino acid sequence) of WO97/09428 and WO97/09429 applications.

SEQ ID NO:38 and 39: 38 kD, the sequence of which is disclosed in SEQ IDNO:154 (DNA) and SEQ ID NO:155 (predicted amino acid sequence) in theU.S. patent application Ser. No. 09/072,967. 38 kD has two alternativeforms, with and without the N-terminal cysteine residue.

SEQ ID NO:40 and 41: DPEP, the sequence of which is disclosed in SEQ IDNO:52 (DNA) and SEQ ID NO:53 (predicted amino acid sequence) in theWO97/09428 and WO97/09429 publications.

SEQ ID NO:42 and 43: TbH4, the sequence of which is disclosed as SEQ IDNO:43 (DNA) and SEQ ID NO:81 (predicted amino acid sequence) inWO97/09428 and WO97/09429 publications.

SEQ ID NO:44 and 45: DPPD, the sequence of which is disclosed in SEQ IDNO:240 (DNA) and SEQ ID NO:241 (predicted amino acid sequence) in U.S.Ser. No. 09/072,967 and in the PCT/US99/03268 and PCT/US99/03265applications. The secreted form of DPPD is shown herein in FIG. 12 ofPCT/US00/28095.

MTb82 (MTb867), the sequence of which is disclosed in FIG. 8 (DNA) and 9(amino acid) of PCT/US00/2809.

Erd14 (MTb16), the cDNA and amino acids sequences of which are disclosedin Verbon et al., J. Bacteriology 174:1352-1359 (1992).

α-crystalline antigen, the sequence of which is disclosed in Verbon etal., J. Bact. 174:1352-1359 (1992);

85 complex antigen, e.g., 85b antigen, the sequence of which isdisclosed in Content et al., Infect. & Immunol. 59:3205-3212 (1991).

The following provides sequences of some additional fusion proteins usedin the compositions and fusion proteins of the invention:

SEQ ID NO:46 and 47: DPV-MTI-MSL-MTCC#2 (MTb71F), the sequence of whichis disclosed as SEQ ID NO:15 (nucleic acid) and in SEQ ID NO:16:(protein) in the U.S. patent application Ser. No. 09/287,849 and in thePCT/US99/07717 application.

SEQ ID NO:48 and 49: DPV-MTI-MSL (MTb31F), the sequence of which isdisclosed in SEQ ID NO:18 (cDNA) and SEQ ID NO:19 (protein) in the U.S.patent application Ser. No. 09/287,849 and in the PCT/US99/07717application.

Each of the above sequences is also disclosed in Cole et al. Nature393:537 (1998) and can be found at, e.g., http://www.sanger.ac.uk andhttp:/www.pasteur.fr/mycdb/.

The above sequences are disclosed in U.S. patent application Ser. Nos.08/523,435, 08/523,436, 08/658,800, 08/659,683, 08/818,111, 08/818,112,08/942,341, 08/942,578, 08/858,998, 08/859,381, 09/056,556, 09/072,596,09/072,967, 09/073,009, 09/073,010, 09/223,040, 09/287,849 09/597,796;and in PCT patent applications PCT/US00/28095; PCT/US98/10407,PCT/US98/10514, PCT/US99/03265, PCT/US99/03268, PCT/US99/07717,WO97/09428 and WO97/09429, WO98/16645, WO98/16646, each of which isherein incorporated by reference.

The antigens described herein include polymorphic variants andconservatively modified variations, as well as inter-strain andinterspecies Mycobacterium homologs. In addition, the antigens describedherein include subsequences or truncated sequences. The fusion proteinsmay also contain additional polypeptides, optionally heterologouspeptides from Mycobacterium or other sources. These antigens may bemodified, for example, by adding linker peptide sequences as describedbelow. These linker peptides may be inserted between one or morepolypeptides which make up each of the fusion proteins.

DEFINITIONS

“Fusion polypeptide” or “fusion protein” refers to a protein having atleast two heterologous Mycobacterium sp. polypeptides covalently linked,either directly or via an amino acid linker. The polypeptides formingthe fusion protein are typically linked C-terminus to N-terminus,although they can also be linked C-terminus to C-terminus, N-terminus toN-terminus, or N-terminus to C-terminus. The polypeptides of the fusionprotein can be in any order. This term also refers to conservativelymodified variants, polymorphic variants, alleles, mutants, subsequences,interspecies homologs, and immunogenic fragments of the antigens thatmake up the fusion protein. Mycobacterium tuberculosis antigens aredescribed in Cole et al., Nature 393:537 (1998), which discloses theentire Mycobacterium tuberculosis genome. The complete sequence ofMycobacterium tuberculosis can also be found at http://www.sanger.ac.ukand at http://www.pasteur.fr/mycdb/ (MycDB). Antigens from otherMycobacterium species that correspond to M. tuberculosis antigens can beidentified, e.g., using sequence comparison algorithms, as describedherein, or other methods known to those of skill in the art, e.g.,hybridization assays and antibody binding assays. Fusion proteins of theinvention can also comprise additional copies of a component antigen orimmunogenic fragment thereof.

A polynucleotide sequence comprising a fusion protein of the inventionhybridizes under stringent conditions to at least two nucleotidesequences, each encoding an antigen polypeptide selected from the groupconsisting of MTB39 or an immunogenic fragment thereof and MTB32A or animmunogenic fragment thereof. The polynucleotide sequences encoding theindividual antigens of the fusion polypeptide therefore includeconservatively modified variants, polymorphic variants, alleles,mutants, subsequences, immunogenic fragments, and interspecies homologsof MTB39 and MTB32A. The polynucleotide sequence encoding the individualpolypeptides of the fusion protein can be in any order.

In some embodiments, the individual polypeptides of the fusion proteinare in order (N- to C-terminus) from large to small. Large antigens areapproximately 30 to 150 kD in size, medium antigens are approximately 10to 30 kD in size, and small antigens are approximately less than 10 kDin size. The sequence encoding the individual polypeptide may be assmall as, e.g., an immunogenic fragment such as an individual CTLepitope encoding about 8 to 9 amino acids, or, e.g., an HTL or B cellepitope. The fragment may also include multiple epitopes. Theimmunogenic fragment may also represent a larger part of the antigensequence, e.g., about 50% or more of MTB39 and MTB32A, e.g., the N- andC-terminal portions of MTB32A. Preferred immunogenic fragments of MTB32Ainclude Ra12, Ra35, and Ra35MutSA.

A fusion polypeptide of the invention specifically binds to antibodiesraised against at least two antigen polypeptides, wherein each antigenpolypeptide is selected from the group consisting of MTB39 or animmunogenic portion or fragment thereof and MTB32A or an immunogenicportion thereof. The antibodies can be polyclonal or monoclonal.Optionally, the fusion polypeptide specifically binds to antibodiesraised against the fusion junction of the antigens, which antibodies donot bind to the antigens individually, i.e., when they are not part of afusion protein. The fusion polypeptides optionally comprise additionalpolypeptides, e.g., three, four, five, six, or more polypeptides, up toabout 25 polypeptides, optionally heterologous polypeptides or repeatedhomologous polypeptides, fused to the at least two heterologousantigens. The additional polypeptides of the fusion protein areoptionally derived from Mycobacterium as well as other sources, such asother bacterial, viral, or invertebrate, vertebrate, or mammaliansources. The individual polypeptides of the fusion protein can be in anyorder. As described herein, the fusion protein can also be linked toother molecules, including additional polypeptides. The compositions ofthe invention can also comprise additional polypeptides that areunlinked to the fusion proteins of the invention. These additionalpolypeptides may be heterologous or homologous polypeptides.

The term “fused” refers to the covalent linkage between two polypeptidesin a fusion protein. The polypeptides are typically joined via a peptidebond, either directly to each other or via an amino acid linker.Optionally, the peptides can be joined via non-peptide covalent linkagesknown to those of skill in the art.

“FL” refers to full-length, i.e., a polypeptide that is the same lengthas the wild-type polypeptide.

The term “immunogenic fragment thereof” refers to a polypeptidecomprising an epitope that is recognized by cytotoxic T lymphocytes,helper T lymphocytes or B cells. Preferred immunogenic fragments of,e.g., MTB32A, are RA35, Ra35MutSA, or Ra12.

The term “Mycobacterium species of the tuberculosis complex” includesthose species traditionally considered as causing the diseasetuberculosis, as well as Mycobacterium environmental and opportunisticspecies that cause tuberculosis and lung disease in immune compromisedpatients, such as patients with AIDS, e.g., M. tuberculosis, M. bovis,or M. africanum, BCG, M. avium, M intracellulare, M. celatum, M.genavense, M. haemophilum, M. kansasii, M. simiae, M. vaccae, M.fortuitum, and M. scrofulaceum (see, e.g., Harrison's Principles ofInternal Medicine, volume 1, pp. 1004-1014 and 1019-1023 (14^(th) ed.,Fauci et al., eds., 1998).

An adjuvant refers to the components in a vaccine or therapeuticcomposition that increase the specific immune response to the antigen(see, e.g., Edelman, AIDS Res. Hum Retroviruses 8:1409-1411 (1992)).Adjuvants induce immune responses of the Th1-type and Th-2 typeresponse. Th1-type cytokines (e.g., IFN-γ, IL-2, and IL-12) tend tofavor the induction of cell-mediated immune response to an administeredantigen, while Th-2 type cytokines (e.g., IL-4, IL-5, 11-6, IL-10 andTNF-β) tend to favor the induction of humoral immune responses.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and 8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins (1984)).

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (e.g., total cellular orlibrary DNA or RNA).

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acid, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditionswill be those in which the salt concentration is less than about 1.0 Msodium ion, typically about 0.01 to 1.0 M sodium ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C. for long probes (e.g., greater than 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide. For selective or specific hybridization, apositive signal is at least two times background, optionally 10 timesbackground hybridization. Exemplary stringent hybridization conditionscan be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42°C., or, 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and0.1% SDS at 65° C.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency.

“Antibody” refers to a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab withpart of the hinge region (see Fundamental Immunology (Paul ed., 3d ed.1993). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990)).

For preparation of monoclonal or polyclonal antibodies, any techniqueknown in the art can be used (see, e.g., Kohler & Milstein, Nature256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Coleet al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy (1985)).Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce antibodies to polypeptides of thisinvention. Also, transgenic mice, or other organisms such as othermammals, may be used to express humanized antibodies. Alternatively,phage display technology can be used to identify antibodies andheteromeric Fab fragments that specifically bind to selected antigens(see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al.,Biotechnology 10:779-783 (1992)).

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein in a heterogeneous population of proteinsand other biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to a particular protein at least two times thebackground and do not substantially bind in a significant amount toother proteins present in the sample. Specific binding to an antibodyunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, polyclonal antibodiesraised to fusion proteins can be selected to obtain only thosepolyclonal antibodies that are specifically immunoreactive with fusionprotein and not with individual components of the fusion proteins. Thisselection may be achieved by subtracting out antibodies that cross-reactwith the individual antigens. A variety of immunoassay formats may beused to select antibodies specifically immunoreactive with a particularprotein. For example, solid-phase ELISA immunoassays are routinely usedto select antibodies specifically immunoreactive with a protein (see,e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988), for adescription of immunoassay formats and conditions that can be used todetermine specific immunoreactivity). Typically a specific or selectivereaction will be at least twice background signal or noise and moretypically more than 10 to 100 times background.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes an individual antigen or a portion thereof) or maycomprise a variant of such a sequence. Polynucleotide variants maycontain one or more substitutions, additions, deletions and/orinsertions such that the biological activity of the encoded fusionpolypeptide is not diminished, relative to a fusion polypeptidecomprising native antigens. Variants preferably exhibit at least about70% identity, more preferably at least about 80% identity and mostpreferably at least about 90% identity to a polynucleotide sequence thatencodes a native polypeptide or a portion thereof.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., 70% identity, optionally 75%, 80%, 85%, 90%, or 95% identity overa specified region), when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection. Such sequences are then said tobe “substantially identical.” This definition also refers to thecompliment of a test sequence. Optionally, the identity exists over aregion that is at least about 25 to about 50 amino acids or nucleotidesin length, or optionally over a region that is 75-100 amino acids ornucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 25 to 500, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendogram showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng & Doolittle, J. Mol. Evol.35:351-360 (1987). The method used is similar to the method described byHiggins & Sharp, CABIOS 5:151-153 (1989). The program can align up to300 sequences, each of a maximum length of 5,000 nucleotides or aminoacids. The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster is then aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences arealigned by a simple extension of the pairwise alignment of twoindividual sequences. The final alignment is achieved by a series ofprogressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. Using PILEUP, a reference sequence is compared to other testsequences to determine the percent sequence identity relationship usingthe following parameters: default gap weight (3.00), default gap lengthweight (0.10), and weighted end gaps. PILEUP can be obtained from theGCG sequence analysis software package, e.g., version 7.0 (Devereaux etal., Nuc. Acids Res. 12:387-395 (1984).

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always>0) and N (penalty score for mismatchingresidues; always<0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

Polynucleotide Compositions

As used herein, the terms “DNA segment” and “polynucleotide” refer to aDNA molecule that has been isolated free of total genomic DNA of aparticular species. Therefore, a DNA segment encoding a polypeptiderefers to a DNA segment that contains one or more coding sequences yetis substantially isolated away from, or purified free from, totalgenomic DNA of the species from which the DNA segment is obtained.Included within the terms “DNA segment” and “polynucleotide” are DNAsegments and smaller fragments of such segments, and also recombinantvectors, including, for example, plasmids, cosmids, phagemids, phage,viruses, and the like.

As will be understood by those skilled in the art, the DNA segments ofthis invention can include genomic sequences, extra-genomic andplasmid-encoded sequences and smaller engineered gene segments thatexpress, or may be adapted to express, proteins, polypeptides, peptidesand the like. Such segments may be naturally isolated, or modifiedsynthetically by the hand of man.

The terms “isolated,” “purified,” or “biologically pure” therefore referto material that is substantially or essentially free from componentsthat normally accompany it as found in its native state. Of course, thisrefers to the DNA segment as originally isolated, and does not excludeother isolated proteins, genes, or coding regions later added to thecomposition by the hand of man. Purity and homogeneity are typicallydetermined using analytical chemistry techniques such as polyacrylamidegel electrophoresis or high performance liquid chromatography. A proteinthat is the predominant species present in a preparation issubstantially purified. An isolated nucleic acid is separated from otheropen reading frames that flank the gene and encode proteins other thanthe gene.

As will be recognized by the skilled artisan, polynucleotides may besingle-stranded (coding or antisense) or double-stranded, and may be DNA(genomic, cDNA or synthetic) or RNA molecules. RNA molecules includeHnRNA molecules, which contain introns and correspond to a DNA moleculein a one-to-one manner, and mRNA molecules, which do not containintrons. Additional coding or non-coding sequences may, but need not, bepresent within a polynucleotide of the present invention, and apolynucleotide may, but need not, be linked to other molecules and/orsupport materials.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes a Mycobacterium antigen or a portion thereof) ormay comprise a variant, or a biological or antigenic functionalequivalent of such a sequence. Polynucleotide variants may contain oneor more substitutions, additions, deletions and/or insertions, asfurther described below, preferably such that the immunogenicity of theencoded polypeptide is not diminished, relative to a native tumorprotein. The effect on the immunogenicity of the encoded polypeptide maygenerally be assessed as described herein. The term “variants” alsoencompasses homologous genes of xenogenic origin.

In additional embodiments, the present invention provides isolatedpolynucleotides and polypeptides comprising various lengths ofcontiguous stretches of sequence identical to or complementary to one ormore of the sequences disclosed herein. For example, polynucleotides areprovided by this invention that comprise at least about 15, 20, 30, 40,50, 75, 100, 150, 200, 300, 400, 500 or 1000 or more contiguousnucleotides of one or more of the sequences disclosed herein as well asall intermediate lengths there between. It will be readily understoodthat “intermediate lengths”, in this context, means any length betweenthe quoted values, such as 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30,31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151,152, 153, etc.; including all integers through 200-500; 500-1,000, andthe like.

The polynucleotides of the present invention, or fragments thereof,regardless of the length of the coding sequence itself, may be combinedwith other DNA sequences, such as promoters, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length may varyconsiderably. It is therefore contemplated that a nucleic acid fragmentof almost any length may be employed, with the total length preferablybeing limited by the ease of preparation and use in the intendedrecombinant DNA protocol. For example, illustrative DNA segments withtotal lengths of about 10,000, about 5000, about 3000, about 2,000,about 1,000, about 500, about 200, about 100, about 50 base pairs inlength, and the like, (including all intermediate lengths) arecontemplated to be useful in many implementations of this invention.

Moreover, it will be appreciated by those of ordinary skill in the artthat, as a result of the degeneracy of the genetic code, there are manynucleotide sequences that encode a polypeptide as described herein. Someof these polynucleotides bear minimal homology to the nucleotidesequence of any native gene. Nonetheless, polynucleotides that vary dueto differences in codon usage are specifically contemplated by thepresent invention, for example polynucleotides that are optimized forhuman and/or primate codon selection. Further, alleles of the genescomprising the polynucleotide sequences provided herein are within thescope of the present invention. Alleles are endogenous genes that arealtered as a result of one or more mutations, such as deletions,additions and/or substitutions of nucleotides. The resulting mRNA andprotein may, but need not, have an altered structure or function.Alleles may be identified using standard techniques (such ashybridization, amplification and/or database sequence comparison).

Polynucleotide Identification and Characterization

Polynucleotides may be identified, prepared and/or manipulated using anyof a variety of well established techniques. For example, apolynucleotide may be identified, as described in more detail below, byscreening a microarray of cDNAs for tumor-associated expression (i.e.,expression that is at least two fold greater in a tumor than in normaltissue, as determined using a representative assay provided herein).Such screens may be performed, for example, using a Synteni microarray(Palo Alto, Calif.) according to the manufacturer's instructions (andessentially as described by Schena et al., Proc. Natl. Acad. Sci. USA93:10614-10619-(1996) and Heller et al: Proc. Natl. Acad. Sci. USA94:2150-2155 (1997)). Alternatively, polynucleotides may be amplifiedfrom cDNA prepared from cells expressing the proteins described herein,such as M. tuberculosis cells. Such polynucleotides may be amplified viapolymerase chain reaction (PCR). For this approach, sequence-specificprimers may be designed based on the sequences provided herein, and maybe purchased or synthesized.

An amplified portion of a polynucleotide of the present invention may beused to isolate a full length gene from a suitable library (e.g., a M.tuberculosis cDNA library) using well known techniques. Within suchtechniques, a library (cDNA or genomic) is screened using one or morepolynucleotide probes or primers suitable for amplification. Preferably,a library is size-selected to include larger molecules. Random primedlibraries may also be preferred for identifying 5′ and upstream regionsof genes. Genomic libraries are preferred for obtaining introns andextending 5′ sequences.

For hybridization techniques, a partial sequence may be labeled (e.g.,by nick-translation or end-labeling with ³²P) using well knowntechniques. A bacterial or bacteriophage library is then generallyscreened by hybridizing filters containing denatured bacterial colonies(or lawns containing phage plaques) with the labeled probe (see Sambrooket al., Molecular Cloning: A Laboratory Manual (1989)). Hybridizingcolonies or plaques are selected and expanded, and the DNA is isolatedfor further analysis. cDNA clones may be analyzed to determine theamount of additional sequence by, for example, PCR using a primer fromthe partial sequence and a primer from the vector. Restriction maps andpartial sequences may be generated to identify one or more overlappingclones. The complete sequence may then be determined using standardtechniques, which may involve generating a series of deletion clones.The resulting overlapping sequences can then assembled into a singlecontiguous sequence. A full length cDNA molecule can be generated byligating suitable fragments, using well known techniques.

Alternatively, there are numerous amplification techniques for obtaininga full length coding sequence from a partial cDNA sequence. Within suchtechniques, amplification is generally performed via PCR. Any of avariety of commercially available kits may be used to perform theamplification step. Primers may be designed using, for example, softwarewell known in the art. Primers are preferably 22-30 nucleotides inlength, have a GC content of at least 50% and anneal to the targetsequence at temperatures of about 68° C. to 72° C. The amplified regionmay be sequenced as described above, and overlapping sequences assembledinto a contiguous sequence.

One such amplification technique is inverse PCR (see Triglia et al.,Nucl. Acids Res. 16:8186 (1988)), which uses restriction enzymes togenerate a fragment in the known region of the gene. The fragment isthen circularized by intramolecular ligation and used as a template forPCR with divergent primers derived from the known region. Within analternative approach, sequences adjacent to a partial sequence may beretrieved by amplification with a primer to a linker sequence and aprimer specific to a known region. The amplified sequences are typicallysubjected to a second round of amplification with the same linker primerand a second primer specific to the known region. A variation on thisprocedure, which employs two primers that initiate extension in oppositedirections from the known sequence, is described in WO 96/38591. Anothersuch technique is known as “rapid amplification of cDNA ends” or RACE.This technique involves the use of an internal primer and an externalprimer, which hybridizes to a polyA region or vector sequence, toidentify sequences that are 5′ and 3′ of a known sequence. Additionaltechniques include capture PCR (Lagerstrom et al., PCR Methods Applic.1:111-19 (1991)) and walking PCR (Parker et al., Nucl. Acids. Res.19:3055-60 (1991)). Other methods employing amplification may also beemployed to obtain a full length cDNA sequence.

In certain instances, it is possible to obtain a full length cDNAsequence by analysis of sequences provided in an expressed sequence tag(EST) database, such as that available from GenBank. Searches foroverlapping ESTs may generally be performed using well known programs(e.g., NCBI BLAST searches), and such ESTs may be used to generate acontiguous full length sequence. Full length DNA sequences may also beobtained by analysis of genomic fragments.

Polynucleotide Expression in Host Cells

In other embodiments of the invention, polynucleotide sequences orfragments thereof which encode polypeptides of the invention, or fusionproteins or functional equivalents thereof, may be used in recombinantDNA molecules to direct expression of a polypeptide in appropriate hostcells. Due to the inherent degeneracy of the genetic code, other DNAsequences that encode substantially the same or a functionallyequivalent amino acid sequence may be produced and these sequences maybe used to clone and express a given polypeptide.

As will be understood by those of skill in the art, it may beadvantageous in some instances to produce polypeptide-encodingnucleotide sequences possessing non-naturally occurring codons. Forexample, codons preferred by a particular prokaryotic or eukaryotic hostcan be selected to increase the rate of protein expression or to producea recombinant RNA transcript having desirable properties, such as ahalf-life which is longer than that of a transcript generated from thenaturally occurring sequence.

Moreover, the polynucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterpolypeptide encoding sequences for a variety of reasons, including butnot limited to, alterations which modify the cloning, processing, and/orexpression of the gene product. For example, DNA shuffling by randomfragmentation and PCR reassembly of gene fragments and syntheticoligonucleotides may be used to engineer the nucleotide sequences. Inaddition, site-directed mutagenesis may be used to insert newrestriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, or introduce mutations, and soforth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences may be ligated to a heterologoussequence to encode a fusion protein. For example, to screen peptidelibraries for inhibitors of polypeptide activity, it may be useful toencode a chimeric protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the polypeptide-encoding sequence and theheterologous protein sequence, so that the polypeptide may be cleavedand purified away from the heterologous moiety.

Sequences encoding a desired polypeptide may be synthesized, in whole orin part, using chemical methods well known in the art (see Caruthers, M.H. et al., Nucl. Acids Res. Symp. Ser. pp. 215-223 (1980), Hom et al.,Nucl. Acids Res. Symp. Ser. pp. 225-232 (1980)). Alternatively, theprotein itself may be produced using chemical methods to synthesize theamino acid sequence of a polypeptide, or a portion thereof. For example,peptide synthesis can be performed using various solid-phase techniques(Roberge et al., Science 269:202-204 (1995)) and automated synthesis maybe achieved, for example, using the ABI 431A Peptide Synthesizer (PerkinElmer, Palo Alto, Calif.).

A newly synthesized peptide may be substantially purified by preparativehigh performance liquid chromatography (e.g., Creighton, Proteins,Structures and Molecular Principles (1983)) or other comparabletechniques available in the art. The composition of the syntheticpeptides may be confirmed by amino acid analysis or sequencing (e.g.,the Edman degradation procedure). Additionally, the amino acid sequenceof a polypeptide, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

In order to express a desired polypeptide, the nucleotide sequencesencoding the polypeptide, or functional equivalents, may be insertedinto appropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence. Methods which are well known to those skilled in theart may be used to construct expression vectors containing sequencesencoding a polypeptide of interest and appropriate transcriptional andtranslational control elements. These methods include in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. Such techniques are described in Sambrook et al.,Molecular Cloning, A Laboratory Manual (1989), and Ausubel et al.,Current Protocols in Molecular Biology (1989).

A variety of expression vector/host systems may be utilized to containand express polynucleotide sequences. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

The “control elements” or “regulatory sequences” present in anexpression vector are those non-translated regions of thevector—enhancers, promoters, 5′ and 3′ untranslated regions—whichinteract with host cellular proteins to carry out transcription andtranslation. Such elements may vary in their strength and specificity.Depending on the vector system and host utilized, any number of suitabletranscription and translation elements, including constitutive andinducible promoters, may be used. For example, when cloning in bacterialsystems, inducible promoters such as the hybrid lacZ promoter of thePBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid(Gibco BRL, Gaithersburg, Md.) and the like may be used. In mammaliancell systems, promoters from mammalian genes or from mammalian virusesare generally preferred. If it is necessary to generate a cell line thatcontains multiple copies of the sequence encoding a polypeptide, vectorsbased on SV40 or EBV may be advantageously used with an appropriateselectable marker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for the expressed polypeptide. Forexample, when large quantities are needed, for example for the inductionof antibodies, vectors which direct high level expression of fusionproteins that are readily purified may be used. Such vectors include,but are not limited to, the multifunctional E. coli cloning andexpression vectors such as BLUESCRIPT (Stratagene), in which thesequence encoding the polypeptide of interest may be ligated into thevector in frame with sequences for the amino-terminal Met and thesubsequent 7 residues of β-galactosidase so that a hybrid protein isproduced; pIN vectors (Van Heeke &Schuster, J. Biol. Chem. 264:5503-5509(1989)); and the like. pGEX Vectors (Promega, Madison, Wis.) may also beused to express foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. Proteins made in such systems may be designed to includeheparin, thrombin, or factor XA protease cleavage sites so that thecloned polypeptide of interest can be released from the GST moiety atwill.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al., Methods Enzymol. 153:516-544 (1987).

In cases where plant expression vectors are used, the expression ofsequences encoding polypeptides may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, EMBO J. 6:307-311 (1987)).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi et al., EMBO J. 3:1671-1680(1984); Broglie et al., Science 224:838-843 (1984); and Winter et al.,Results Probl. Cell. Differ. 17:85-105 (1991)). These constructs can beintroduced into plant cells by direct DNA transformation orpathogen-mediated transfection. Such techniques are described in anumber of generally available reviews (see, e.g., Hobbs in McGraw HillYearbook of Science and Technology pp. 191-196 (1992)).

An insect system may also be used to express a polypeptide of interest.For example, in one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia larvae. The sequencesencoding the polypeptide may be cloned into a non-essential region ofthe virus, such as the polyhedrin gene, and placed under control of thepolyhedrin promoter. Successful insertion of the polypeptide-encodingsequence will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which the polypeptide of interest may be expressed (Engelhardet al., Proc. Natl. Acad. Sci. U.S.A. 91:3224-3227 (1994)).

In mammalian host cells, a number of viral-based expression systems aregenerally available. For example, in cases where an adenovirus is usedas an expression vector, sequences encoding a polypeptide of interestmay be ligated into an adenovirus transcription/translation complexconsisting of the late promoter and tripartite leader sequence.Insertion in a non-essential E1 or E3 region of the viral genome may beused to obtain a viable virus which is capable of expressing thepolypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad.Sci. U.S.A. 81:3655-3659 (1984)). In addition, transcription, enhancers,such as the Rous sarcoma virus (RSV) enhancer, may be used to increaseexpression in mammalian host cells.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding a polypeptide of interest. Suchsignals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding the polypeptide, its initiation codon,and upstream sequences are inserted into the appropriate expressionvector, no additional transcriptional or translational control signalsmay be needed. However, in cases where only coding sequence, or aportion thereof, is inserted, exogenous translational control signalsincluding the ATG initiation codon should be provided. Furthermore, theinitiation codon should be in the correct reading frame to ensuretranslation of the entire insert. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers which are appropriate for the particular cell system which isused, such as those described in the literature (Scharf. et al., ResultsProbl. Cell Differ. 20:125-162 (1994)).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation.glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, andW138, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is generally preferred. For example, cell lines which stablyexpress a polynucleotide of interest may be transformed using expressionvectors which may contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same or on aseparate vector. Following the introduction of the vector, cells may beallowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells which successfully express the introduced sequences.Resistant clones of stably transformed cells may be proliferated usingtissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler et al., Cell 11:223-32 (1977)) and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817-23 (1990)) geneswhich can be employed in tk.sup.- or aprt.sup.-cells, respectively.Also, antimetabolite, antibiotic or herbicide resistance can be used asthe basis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler et al., Proc. Natl. Acad. Sci. U.S.A. 77:3567-70(1980)); npt, which confers resistance to the aminoglycosides, neomycinand G-418 (Colbere-Garapin et al., J. Mol. Biol. 150:1-14 (1981)); andals or pat, which confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively (Murry, supra). Additional selectablegenes have been described, for example, trpB, which allows cells toutilize indole in place of tryptophan, or hisD, which allows cells toutilize histinol in place of histidine (Hartman & Mulligan, Proc. Natl.Acad. Sci. U.S.A. 85:8047-51 (1988)). Recently, the use of visiblemarkers has gained popularity with such markers as anthocyanins,β-glucuronidase and its substrate GUS, and luciferase and its substrateluciferin, being widely used not only to identify transformants, butalso to quantify the amount of transient or stable protein expressionattributable to a specific vector system (Rhodes et al., Methods Mol.Biol. 55:121-131 (1995)).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding apolypeptide is inserted within a marker gene sequence, recombinant cellscontaining sequences can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with apolypeptide-encoding sequence under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

Alternatively, host cells which contain and express a desiredpolynucleotide sequence may be identified by a variety of proceduresknown to those of skill in the art. These procedures include, but arenot limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassayor immunoassay techniques which include membrane, solution, or chipbased technologies for the detection and/or quantification of nucleicacid or protein.

A variety of protocols for detecting and measuring the expression ofpolynucleotide-encoded products, using either polyclonal or monoclonalantibodies specific for the product are known in the art. Examplesinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA), and fluorescence activated cell sorting (FACS). A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering epitopes on a given polypeptide may be preferred forsome applications, but a competitive binding assay may also be employed.These and other assays are described, among other places, in Hampton etal., Serological Methods, a Laboratory Manual (1990) and Maddox et al.,J. Exp. Med. 158:1211-1216 (1983).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides include oligolabeling,nick translation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the sequences, or any portions thereof may becloned into a vector for the production of an mRNA probe. Such vectorsare known in the art, are commercially available, and may be used tosynthesize RNA probes in vitro by addition of an appropriate RNApolymerase such as T7, T3, or SP6 and labeled nucleotides. Theseprocedures may be conducted using a variety of commercially availablekits. Suitable reporter molecules or labels, which may be used includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

Host cells transformed with a polynucleotide sequence of interest may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a recombinantcell may be secreted or contained intracellularly depending on thesequence and/or the vector used: As will be understood by those of skillin the art, expression vectors containing polynucleotides of theinvention may be designed to contain signal sequences which directsecretion of the encoded polypeptide through a prokaryotic or eukaryoticcell membrane. Other recombinant constructions may be used to joinsequences encoding a polypeptide of interest to nucleotide sequenceencoding a polypeptide domain which will facilitate purification ofsoluble proteins. Such purification facilitating domains include, butare not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen. San Diego, Calif.) between the purificationdomain and the encoded polypeptide may be used to facilitatepurification. One such expression vector provides for expression of afusion protein containing a polypeptide of interest and a nucleic acidencoding 6 histidine residues preceding a thioredoxin or an enterokinasecleavage site. The histidine residues facilitate purification on IMIAC(immobilized metal ion affinity chromatography) as described in Porathet al., Prot. Exp. Purif. 3:263-281 (1992) while the enterokinasecleavage site provides a means for purifying the desired polypeptidefrom the fusion protein. A discussion of vectors which contain fusionproteins is provided in Kroll et al., DNA Cell Biol. 12:441-453 (1993)).

In addition to recombinant production methods, polypeptides of theinvention, and fragments thereof, may be produced by direct peptidesynthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc.85:2149-2154 (1963)). Protein synthesis may be performed using manualtechniques or by automation. Automated synthesis may be achieved, forexample, using Applied Biosystems 431 A Peptide Synthesizer (PerkinElmer). Alternatively, various fragments may be chemically synthesizedseparately and combined using chemical methods to produce the fulllength molecule.

In Vivo Polynucleotide Delivery Techniques

In additional embodiments, genetic constructs comprising one or more ofthe polynucleotides of the invention are introduced into cells in vivo.This may be achieved using any of a variety or well known approaches,several of which are outlined below for the purpose of illustration.

1. Adenovirus

One of the preferred methods for in vivo delivery of one or more nucleicacid sequences involves the use of an adenovirus expression vector.“Adenovirus expression vector” is meant to include those constructscontaining adenovirus sequences sufficient to (a) support packaging ofthe construct and (b) to express a polynucleotide that has been clonedtherein in a sense or antisense orientation. Of course, in the contextof an antisense construct, expression does not require that the geneproduct be synthesized.

The expression vector comprises a genetically engineered form of anadenovirus. Knowledge of the genetic organization of adenovirus, a 36kb, linear, double-stranded DNA virus, allows substitution of largepieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus &Horwitz, 1992). In contrast to retrovirus, the adenoviral infection ofhost cells does not result in chromosomal integration because adenoviralDNA can replicate in an episomal manner without potential genotoxicity.Also, adenoviruses are structurally stable, and no genome rearrangementhas been detected after extensive amplification. Adenovirus can infectvirtually all epithelial cells regardless of their cell cycle stage. Sofar, adenoviral infection appears to be linked only to mild disease suchas acute respiratory disease in humans.

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized genome, ease of manipulation, high titer, widetarget-cell range and high infectivity. Both ends of the viral genomecontain 100-200 base pair inverted repeats (ITRs), which are ciselements necessary for viral DNA replication and packaging. The early(E) and late (L) regions of the genome contain different transcriptionunits that are divided by the onset of viral DNA replication. The E1region (EIA and EIB) encodes proteins responsible for the regulation oftranscription of the viral genome and a few cellular genes. Theexpression of the E2 region (E2A and E2B) results in the synthesis ofthe proteins for viral DNA replication. These proteins are involved inDNA replication, late gene expression and host cell shut-off (Renan,1990). The products of the late genes, including the majority of theviral capsid proteins, are expressed only after significant processingof a single primary transcript issued by the major late promoter (MLP).The MLP, (located at 16.8 m.u.) is particularly efficient during thelate phase of infection, and all the mRNA's issued from this promoterpossess a 5′-tripartite leader (TPL) sequence which makes them preferredmRNA's for translation.

In a current system, recombinant adenovirus is generated from homologousrecombination between shuttle vector and provirus vector. Due to thepossible recombination between two proviral vectors, wild-typeadenovirus may be generated from this process. Therefore, it is criticalto isolate a single clone of virus from an individual plaque and examineits genomic structure.

Generation and propagation of the current adenovirus vectors, which arereplication deficient, depend on a unique helper cell line, designated293, which was transformed from human embryonic kidney cells by Ad5 DNAfragments and constitutively expresses E1 proteins (Graham et al.,1977). Since the E3 region is dispensable from the adenovirus genome(Jones & Shenk, 1978), the current adenovirus vectors, with the help of293 cells, carry foreign DNA in either the E1, the D3 or both regions(Graham & Prevec, 1991). In nature, adenovirus can package approximately105% of the wild-type genome (Ghosh-Choudhury et al., 1987), providingcapacity for about 2 extra kB of DNA. Combined with the approximately5.5 kB of DNA that is replaceable in the E1 and E3 regions, the maximumcapacity of the current adenovirus vector is under 7.5 kB, or about 15%of the total length of the vector. More than 80% of the adenovirus viralgenome remains in the vector backbone and is the source of vector-bornecytotoxicity. Also, the replication deficiency of the E1-deleted virusis incomplete. For example, leakage of viral gene expression has beenobserved with the currently available vectors at high multiplicities ofinfection (MOI) (Mulligan, 1993).

Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. As stated above,the currently preferred helper cell line is 293.

Recently, Racher et al. (1995) disclosed improved methods for culturing293 cells and propagating adenovirus. In one format, natural cellaggregates are grown by inoculating individual cells into 1 litersiliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 mlof medium. Following stirring at 40 rpm, the cell viability is estimatedwith trypan blue. In another format, Fibra-Cel microcarriers (BibbySterlin, Stone, UK) (5 g/l) is employed as follows. A cell inoculum,resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250ml Erlenmeyer flask and left stationary, with occasional agitation, for1 to 4 h. The medium is then replaced with 50 ml of fresh medium andshaking initiated. For virus production, cells are allowed to grow toabout 80% confluence, after which time the medium is replaced (to 25% ofthe final volume) and adenovirus added at an MOI of 0.05. Cultures areleft stationary overnight, following which the volume is increased to100% and shaking commenced for another 72 h.

Other than the requirement that the adenovirus vector be replicationdefective, or at least conditionally defective, the nature of theadenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain aconditional replication-defective adenovirus vector for use in thepresent invention, since Adenovirus type 5 is a human adenovirus aboutwhich a great deal of biochemical and genetic information is known, andit has historically been used for most constructions employingadenovirus as a vector.

As stated above, the typical vector according to the present inventionis replication defective and will not have an adenovirus E1 region.Thus, it will be most convenient to introduce the polynucleotideencoding the gene of interest at the position from which the E1-codingsequences have been removed. However, the position of insertion of theconstruct within the adenovirus sequences is not critical to theinvention. The polynucleotide encoding the gene of interest may also beinserted in lieu of the deleted E3 region in E3 replacement vectors asdescribed by Karlsson et al. (1986) or in the E4 region where a helpercell line or helper virus complements the E4 defect.

Adenovirus is easy to grow and manipulate and exhibits broad host rangein vitro and in vivo. This group of viruses can be obtained in hightiters, e.g., 109-10¹¹ plaque-forming units per ml, and they are highlyinfective. The life cycle of adenovirus does not require integrationinto the host cell genome. The foreign genes delivered by adenovirusvectors are episomal and, therefore, have low genotoxicity to hostcells. No side effects have been reported in studies of vaccination withwild-type adenovirus (Couch et al., 1963; Top et al., 1971),demonstrating their safety and therapeutic potential as in vivo genetransfer vectors.

Adenovirus vectors have been used in eukaryotic gene expression (Levreroet al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhaus& Horwitz, 1992; Graham & Prevec, 1992). Recently, animal studiessuggested that recombinant adenovirus could be used for gene therapy(Stratford-Perricaudet & Perricaudet, 1991; Stratford-Penicaudet et al.,1990; Rich et al., 1993). Studies in administering recombinantadenovirus to different tissues include trachea instillation (Rosenfeldet al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al.,1993), peripheral intravenous injections (Herz & Gerard, 1993) andstereotactic inoculation into the brain (Le Gal La Salle et al., 1993).

2. Retroviruses

The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants. The retroviral genome contains three genes,gag, pol, and env that code for capsid proteins, polymerase enzyme, andenvelope components, respectively. A sequence found upstream from thegag gene contains a signal for packaging of the genome into virions. Twolong terminal repeat (LTR) sequences are present at the 5′ and 3′ endsof the viral genome. These contain strong promoter and enhancersequences and are also required for integration in the host cell genome(Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding oneor more oligonucleotide or polynucleotide sequences of interest isinserted into the viral genome in the place of certain viral sequencesto produce a virus that is replication-defective. In order to producevirions, a packaging cell line containing the gag, pol, and env genesbut without the LTR and packaging components is constructed (Mann etal., 1983). When a recombinant plasmid containing a cDNA, together withthe retroviral LTR and packaging sequences is introduced into this cellline (by calcium phosphate precipitation for example), the packagingsequence allows the RNA transcript of the recombinant plasmid to bepackaged into viral particles, which are then secreted into the culturemedia (Nicolas & Rubenstein, 1988; Temin, 1986; Mann et al., 1983). Themedia containing the recombinant retroviruses is then collected,optionally concentrated, and used for gene transfer. Retroviral vectorsare able to infect a broad variety of cell types. However, integrationand stable expression require the division of host cells (Paskind etal., 1975).

A novel approach designed to allow specific targeting of retrovirusvectors was recently developed based on the chemical modification of aretrovirus by the chemical addition of lactose residues to the viralenvelope. This modification could permit the specific infection ofhepatocytes via sialoglycoprotein receptors.

A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin (Roux etal., 1989). Using antibodies against major histocompatibility complexclass I and class II antigens, they demonstrated the infection of avariety of human cells that bore those surface antigens with anecotropic virus in vitro (Roux et al., 1989).

3. Adeno-Associated Viruses

AAV (Ridgeway, 1988; Hermonat & Muzycska, 1984) is a parovirus,discovered as a contamination of adenoviral stocks. It is a ubiquitousvirus (antibodies are present in 85% of the US human population) thathas not been linked to any disease. It is also classified as adependovirus, because its replications is dependent on the presence of ahelper virus, such as adenovirus. Five serotypes have been isolated, ofwhich AAV-2 is the best characterized. AAV has a single-stranded linearDNA that is encapsidated into capsid proteins VP1, VP2 and VP3 to forman icosahedral virion of 20 to 24 nm in diameter (Muzyczka & McLaughlin,1988).

The AAV DNA is approximately 4.7 kilobases long. It contains two openreading frames and is flanked by two ITRs. There are two major genes inthe AAV genome: rep and cap. The rep gene codes for proteins responsiblefor viral replications, whereas cap codes for capsid protein VP1-3. EachITR forms a T-shaped hairpin structure. These terminal repeats are theonly essential cis components of the AAV for chromosomal integration.Therefore, the AAV can be used as a vector with all viral codingsequences removed and replaced by the cassette of genes for delivery.Three viral promoters have been identified and named p5, p19, and p40,according to their map position. Transcription from p5 and p19 resultsin production of rep proteins, and transcription from p40 produces thecapsid proteins (Hermonat & Muzyczka, 1984).

There are several factors that prompted researchers to study thepossibility of using rAAV as an expression vector One is that therequirements for delivering a gene to integrate into the host chromosomeare surprisingly few. It is necessary to have the 145-bp ITRs, which areonly 6% of the AAV genome. This leaves room in the vector to assemble a4.5-kb DNA insertion. While this carrying capacity may prevent the AAVfrom delivering large genes, it is amply suited for delivering theantisense constructs of the present invention.

AAV is also a good choice of delivery vehicles due to its safety. Thereis a relatively complicated rescue mechanism: not only wild typeadenovirus but also AAV genes are required to mobilize rAAV. Likewise,AAV is not pathogenic and not associated with any disease. The removalof viral coding sequences minimizes immune reactions to viral geneexpression, and therefore, rAAV does not evoke an inflammatory response.

4. Other Viral Vectors as Expression Constructs

Other viral vectors may be employed as expression constructs in thepresent invention for the delivery of oligonucleotide or polynucleotidesequences to a host cell. Vectors derived from viruses such as vacciniavirus (Ridgeway, 1988; Coupar et al., 1988), lentiviruses, polio virusesand herpes viruses may be employed. They offer several attractivefeatures for various mammalian cells (Friedmann, 1989; Ridgeway, 1988;Coupar et al., 1988; Horwich et al., 1990).

With the recent recognition of defective hepatitis B viruses, newinsight was gained into the structure-function relationship of differentviral sequences. In vitro studies showed that the virus could retain theability for helper-dependent packaging and reverse transcription despitethe deletion of up to 80% of its genome (Horwich et al., 1990). Thissuggested that large portions of the genome could be replaced withforeign genetic material. The hepatotropism and persistence(integration) were particularly attractive properties for liver-directedgene transfer. Chang et al. (1991) introduced the chloramphenicolacetyltransferase (CAT) gene into duck hepatitis B virus genome in theplace of the polymerase, surface, and pre-surface coding sequences. Itwas cotransfected with wild-type virus into an avian hepatoma cell line.Culture media containing high titers of the recombinant virus were usedto infect primary duckling hepatocytes. Stable CAT gene expression wasdetected for at least 24 days after transfection (Chang et al., 1991).

5. Non-Viral Vectors

In order to effect expression of the oligonucleotide or polynucleotidesequences of the present invention, the expression construct must bedelivered into a cell. This delivery may be accomplished in vitro, as inlaboratory procedures for transforming cells lines, or in vivo or exvivo, as in the treatment of certain disease states. As described above,one preferred mechanism for delivery is via viral infection where theexpression construct is encapsulated in an infectious viral particle.

Once the expression construct has been delivered into the cell thenucleic acid encoding the desired oligonucleotide or polynucleotidesequences may be positioned and expressed at different sites. In certainembodiments, the nucleic acid encoding the construct may be stablyintegrated into the genome of the cell. This integration may be in thespecific location and orientation via homologous recombination (genereplacement) or it may be integrated in a random, non-specific location(gene augmentation). In yet further embodiments, the nucleic acid may bestably maintained in the cell as a separate, episomal segment of DNA.Such nucleic acid segments or “episomes” encode sequences sufficient topermit maintenance and replication independent of or in synchronizationwith the host cell cycle. How the expression construct is delivered to acell and where in the cell the nucleic acid remains is dependent on thetype of expression construct employed.

In certain embodiments of the invention, the expression constructcomprising one or more oligonucleotide or polynucleotide sequences maysimply consist of naked recombinant DNA or plasmids. Transfer of theconstruct may be performed by any of the methods mentioned above whichphysically or chemically permeabilize the cell membrane. This isparticularly applicable for transfer in vitro but it may be applied toin vivo use as well. Dubensky et al. (1984) successfully injectedpolyomavirus DNA in the form of calcium phosphate precipitates intoliver and spleen of adult and newborn mice demonstrating active viralreplication and acute infection. Benvenisty & Reshef (1986) alsodemonstrated that direct intraperitoneal injection of calciumphosphate-precipitated plasmids results in expression of the transfectedgenes. It is envisioned that DNA encoding a gene of interest may also betransferred in a similar manner in vivo and express the gene product.

Another embodiment of the invention for transferring a naked DNAexpression construct into cells may involve particle bombardment. Thismethod depends on the ability to accelerate DNA-coated microprojectilesto a high velocity allowing them to pierce cell membranes and entercells without killing them (Klein et al., 1987). Several devices foraccelerating small particles have been developed. One such device relieson a high voltage discharge to generate an electrical current, which inturn provides the motive force (Yang et al., 1990). The microprojectilesused have consisted of biologically inert substances such as tungsten orgold beads.

Selected organs including the liver, skin, and muscle tissue of rats andmice have been bombarded in vivo (Yang et al., 1990; Zelenin et al.,1991). This may require surgical exposure of the tissue or cells, toeliminate any intervening tissue between the gun and the target organ,i.e., ex vivo treatment. Again, DNA encoding a particular gene may bedelivered via this method and still be incorporated by the presentinvention.

Polypeptide Compositions

The present invention, in other aspects, provides polypeptidecompositions. Generally, a polypeptide of the invention will be anisolated polypeptide (or an epitope, variant, or active fragmentthereof) derived from a mammalian species. Preferably, the polypeptideis encoded by a polynucleotide sequence disclosed herein or a sequencewhich hybridizes under moderately stringent conditions to apolynucleotide sequence disclosed herein. Alternatively, the polypeptidemay be defined as a polypeptide which comprises a contiguous amino acidsequence from an amino acid sequence disclosed herein, or whichpolypeptide comprises an entire amino acid sequence disclosed herein.

Immunogenic portions may generally be identified using well knowntechniques, such as those summarized in Paul, Fundamental Immunology,3rd ed., 243-247 (1993) and references cited therein. Such techniquesinclude screening polypeptides for the ability to react withantigen-specific antibodies, antisera and/or T-cell lines or clones. Asused herein, antisera and antibodies are “antigen-specific” if theyspecifically bind to an antigen (i.e., they react with the protein in anELISA or other immunoassay, and do not react detectably with unrelatedproteins). Such antisera and antibodies may be prepared as describedherein, and using well known techniques. An immunogenic portion of aMycobacterium sp. protein is a portion that reacts with such antiseraand/or T-cells at a level that is not substantially less than thereactivity of the full length polypeptide (e.g., in an ELISA and/orT-cell reactivity assay). Such immunogenic portions may react withinsuch assays at a level that is similar to or greater than the reactivityof the full length polypeptide. Such screens may generally be performedusing methods well known to those of ordinary skill in the art, such asthose described in Harlow & Lane, Antibodies: A Laboratory Manual(1988). For example, a polypeptide may be immobilized on a solid supportand contacted with patient sera to allow binding of antibodies withinthe sera to the immobilized polypeptide. Unbound sera may then beremoved and bound antibodies detected using, for example, ¹²⁵I-labeledProtein A.

Polypeptides may be prepared using any of a variety of well knowntechniques. Recombinant polypeptides encoded by DNA sequences asdescribed above may be readily prepared from the DNA sequences using anyof a variety of expression vectors known to those of ordinary skill inthe art. Expression may be achieved in any appropriate host cell thathas been transformed or transfected with an expression vector containinga DNA molecule that encodes a recombinant polypeptide. Suitable hostcells include prokaryotes, yeast, and higher eukaryotic cells, such asmammalian cells and plant cells. Preferably, the host cells employed areE. coli, yeast or a mammalian cell line such as COS or CHO. Supernatantsfrom suitable host/vector systems which secrete recombinant protein orpolypeptide into culture media may be first concentrated using acommercially available filter. Following concentration, the concentratemay be applied to a suitable purification matrix such as an affinitymatrix or an ion exchange resin. Finally, one or more reverse phase HPLCsteps can be employed to further purify a recombinant polypeptide.

Polypeptides of the invention, immunogenic fragments thereof, and othervariants having less than about 100 amino acids, and generally less thanabout 50 amino acids, may also be generated by synthetic means, usingtechniques well known to those of ordinary skill in the art. Forexample, such polypeptides may be synthesized using any of thecommercially available solid-phase techniques, such as the Merrifieldsolid-phase synthesis method, where amino acids are sequentially addedto a growing amino acid chain. See Merrifield, J. Am. Chem. Soc.85:2149-2146 (1963). Equipment for automated synthesis of polypeptidesis commercially available from suppliers such as Perkin Elmer/AppliedBioSystems Division (Foster City, Calif.), and may be operated accordingto the manufacturer's instructions.

Within certain specific embodiments, a polypeptide may be a fusionprotein that comprises multiple polypeptides as described herein, orthat comprises at least one polypeptide as described herein and anunrelated sequence, such as a known tumor protein. A fusion partner may,for example, assist in providing T helper epitopes (an immunologicalfusion partner), preferably T helper epitopes recognized by humans, ormay assist in expressing the protein (an expression enhancer) at higheryields than the native recombinant protein. Certain preferred fusionpartners are both immunological and expression enhancing fusionpartners. Other fusion partners may be selected so as to increase thesolubility of the protein or to enable the protein to be targeted todesired intracellular compartments. Still further fusion partnersinclude affinity tags, which facilitate purification of the protein.

Fusion proteins may generally be prepared using standard techniques,including chemical conjugation. Preferably, a fusion protein isexpressed as a recombinant protein, allowing the production of increasedlevels, relative to a non-fused protein, in an expression system.Briefly, DNA sequences encoding the polypeptide components may beassembled separately, and ligated into an appropriate expression vector.The 3′ end of the DNA sequence encoding one polypeptide component isligated, with or without a peptide linker, to the 5′ end of a DNAsequence encoding the second polypeptide component so that the readingframes of the sequences are in phase. This permits translation into asingle fusion protein that retains the biological activity of bothcomponent polypeptides.

A peptide linker sequence may be employed to separate the first andsecond polypeptide components by a distance sufficient to ensure thateach polypeptide folds into its secondary and tertiary structures. Sucha peptide linker sequence is incorporated into the fusion protein usingstandard techniques well known in the art. Suitable peptide linkersequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Preferred peptide linker sequences contain Gly, Asnand Ser residues. Other near neutral amino acids, such as Thr and Alamay also be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Maratea etal., Gene 40:39-46 (1985); Murphy et al., Proc. Natl. Acad. Sci. USA83:8258-8262 (1986); U.S. Pat. No. 4,935,233 and U.S. Pat. No.4,751,180. The linker sequence may generally be from 1 to about 50 aminoacids in length. Linker sequences are not required when the first andsecond polypeptides have non-essential N-terminal amino acid regionsthat can be used to separate the functional domains and prevent stericinterference.

The ligated DNA sequences are operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements responsible for expression of DNA are located only 5′ to theDNA sequence encoding the first polypeptides. Similarly, stop codonsrequired to end translation and transcription termination signals areonly present 3′ to the DNA sequence encoding the second polypeptide.

Fusion proteins are also provided. Such proteins comprise a polypeptideas described herein together with an unrelated immunogenic protein.Preferably the immunogenic protein is capable of eliciting a recallresponse. Examples of such proteins include tetanus, tuberculosis andhepatitis proteins (see, e.g., Stoute et al., New Engl. J. Med.336:86-91 (1997)).

Within preferred embodiments, an immunological fusion partner is derivedfrom protein D, a surface protein of the gram-negative bacteriumHaemophilus influenza B (WO 91/18926). Preferably, a protein Dderivative comprises approximately the first third of the protein (e.g.,the first N-terminal 100-110 amino acids), and a protein D derivativemay be lipidated. Within certain preferred embodiments, the first 109residues of a lipoprotein D fusion partner is included on the N-terminusto provide the polypeptide with additional exogenous T-cell epitopes andto increase the expression level in E. coli (thus functioning as anexpression enhancer). The lipid tail ensures optimal presentation of theantigen to antigen presenting cells. Other fusion partners include thenon-structural protein from influenzae virus, NS1 (hemaglutinin).Typically, the N-terminal 81 amino acids are used, although differentfragments that include T-helper epitopes may be used.

In another embodiment, the immunological fusion partner is the proteinknown as LYTA, or a portion thereof (preferably a C-terminal portion).LYTA is derived from Streptococcus pneumoniae, which synthesizes anN-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytAgene; Gene 43:265-292 (1986)). LYTA is an autolysin that specificallydegrades certain bonds in the peptidoglycan backbone. The C-terminaldomain of the LYTA protein is responsible for the affinity to thecholine or to some choline analogues such as DEAE. This property hasbeen exploited for the development of E. coli C-LYTA expressing plasmidsuseful for expression of fusion proteins. Purification of hybridproteins containing the C-LYTA fragment at the amino terminus has beendescribed (see Biotechnology 10:795-798 (1992)). Within a preferredembodiment, a repeat portion of LYTA may be incorporated into a fusionprotein. A repeat portion is found in the C-terminal region starting atresidue 178. A particularly preferred repeat portion incorporatesresidues 188-305.

In general, polypeptides (including fusion proteins) and polynucleotidesas described herein are isolated. An “isolated” polypeptide orpolynucleotide is one that is removed from its original environment. Forexample, a naturally-occurring protein is isolated if it is separatedfrom some or all of the coexisting materials in the natural system.Preferably, such polypeptides are at least about 90% pure, morepreferably at least about 95% pure and most preferably at least about99% pure. A polynucleotide is considered to be isolated if, for example,it is cloned into a vector that is not a part of the naturalenvironment.

T Cells

Immunotherapeutic compositions may also, or alternatively, comprise Tcells specific for a Mycobacterium antigen. Such cells may generally beprepared in vitro or ex vivo, using standard procedures. For example, Tcells may be isolated from bone marrow, peripheral blood, or a fractionof bone marrow or peripheral blood of a patient, using a commerciallyavailable cell separation system, such as the Isolex™ System, availablefrom Nexell Therapeutics, Inc. (Irvine, Calif.; see also U.S. Pat. No.5,240,856; U.S. Pat. No. 5,215,926; WO 89/06280; WO 91/16116 and WO92/07243). Alternatively, T cells may be derived from related orunrelated humans, non-human mammals, cell lines or cultures.

T cells may be stimulated with a polypeptide of the invention,polynucleotide encoding such a polypeptide, and/or an antigen presentingcell (APC) that expresses such a polypeptide. Such stimulation isperformed under conditions and for a time sufficient to permit thegeneration of T cells that are specific for the polypeptide. Preferably,the polypeptide or polynucleotide is present within a delivery vehicle,such as a microsphere, to facilitate the generation of specific T cells.

T cells are considered to be specific for a polypeptide of the inventionif the T cells specifically proliferate, secrete cytokines or killtarget cells coated with the polypeptide or expressing a gene encodingthe polypeptide. T cell specificity may be evaluated using any of avariety of standard techniques. For example, within a chromium releaseassay or proliferation assay, a stimulation index of more than two foldincrease in lysis and/or proliferation, compared to negative controls,indicates T cell specificity. Such assays may be performed, for example,as described in Chen et al., Cancer Res. 54:1065-1070 (1994)).Alternatively, detection of the proliferation of T cells may beaccomplished by a variety of known techniques. For example, T cellproliferation can be detected by measuring an increased rate of DNAsynthesis (e.g., by pulse-labeling cultures of T cells with tritiatedthymidine and measuring the amount of tritiated thymidine incorporatedinto DNA). Contact with a polypeptide of the invention (100 ng/ml-100μg/ml, preferably 200 ng/ml-25 μg/ml) for 3-7 days should result in atleast a two fold increase in proliferation of the T cells. Contact asdescribed above for 2-3 hours should result in activation of the Tcells, as measured using standard cytokine assays in which a two foldincrease in the level of cytokine release (e.g., TNF or IFN-γ) isindicative of T cell activation (see Coligan et al., Current Protocolsin Immunology, vol. 1 (1998)). T cells that have been activated inresponse to a polypeptide, polynucleotide or polypeptide-expressing APCmay be CD4⁺ and/or CD8⁺. Protein-specific T cells may be expanded usingstandard techniques. Within preferred embodiments, the T cells arederived from a patient, a related donor or an unrelated donor, and areadministered to the patient following stimulation and expansion.

For therapeutic purposes, CD4⁺ or CD8⁺ T cells that proliferate inresponse to a polypeptide, polynucleotide or APC can be expanded innumber either in vitro or in vivo. Proliferation of such T cells invitro may be accomplished in a variety of ways. For example, the T cellscan be re-exposed to a polypeptide, or a short peptide corresponding toan immunogenic portion of such a polypeptide, with or without theaddition of T cell growth factors, such as interleukin-2, and/orstimulator cells that synthesize the polypeptide. Alternatively, one ormore T cells that proliferate in the presence of the protein can beexpanded in number by cloning. Methods for cloning cells are well knownin the art, and include limiting dilution.

Pharmaceutical Compositions

In additional embodiments, the present invention concerns formulation ofone or more of the polynucleotide, polypeptide, T-cell and/or antibodycompositions disclosed herein in pharmaceutically-acceptable orphysiologically-acceptable solutions for administration to a cell or ananimal, either alone, or in combination with one or more othermodalities of therapy. Such compositions are also useful for diagnosticuses.

It will also be understood that, if desired, the nucleic acid segment,RNA, DNA or PNA compositions that express a polypeptide as disclosedherein may be administered in combination with other agents as well,such as, e.g., other proteins or polypeptides or variouspharmaceutically-active agents. In fact, there is virtually no limit toother components that may also be included, given that the additionalagents do not cause a significant adverse effect upon contact with thetarget cells or host tissues. The compositions may thus be deliveredalong with various other agents as required in the particular instance.Such compositions may be purified from host cells or other biologicalsources, or alternatively may be chemically synthesized as describedherein. Likewise, such compositions may further comprise substituted orderivatized RNA or DNA compositions.

Formulation of pharmaceutically-acceptable excipients and carriersolutions is well-known to those of skill in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., oral, parenteral, intravenous, intranasal, andintramuscular administration and formulation.

1. Oral Delivery

In certain applications, the pharmaceutical compositions disclosedherein may be delivered via oral administration to an animal. As such,these compositions may be formulated with an inert diluent or with anassimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

The active compounds may even be incorporated with excipients and usedin the form of ingestible tablets, buccal tables, troches, capsules,elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et al.,1997; Hwang et al., 1998; U.S. Pat. No. 5,641,515; U.S. Pat. No.5,580,579 and U.S. Pat. No. 5,792,451, each specifically incorporatedherein by reference in its entirety). The tablets, troches, pills,capsules and the like may also contain the following: a binder, as gumtragacanth, acacia, cornstarch, or gelatin; excipients, such asdicalcium phosphate; a disintegrating agent, such as corn starch, potatostarch, alginic acid and the like; a lubricant, such as magnesiumstearate; and a sweetening agent, such as sucrose, lactose or saccharinmay be added or a flavoring agent, such as peppermint, oil ofwintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar, or both.A syrup of elixir may contain the active compound sucrose as asweetening agent methyl and propylparabens as preservatives, a dye andflavoring, such as cherry or orange flavor. Of course, any material usedin preparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, the activecompounds may be incorporated into sustained-release preparation andformulations.

Typically, these formulations may contain at least about 0.1% of theactive compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 60% or 70% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound(s) ineach therapeutically useful composition may be prepared is such a waythat a suitable dosage will be obtained in any given unit dose of thecompound. Factors such as solubility, bioavailability, biologicalhalf-life, route of administration, product shelf life, as well as otherpharmacological considerations will be contemplated by one skilled inthe art of preparing such pharmaceutical formulations, and as such, avariety of dosages and treatment regimens may be desirable.

For oral administration the compositions of the present invention mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation. For example, a mouthwash may beprepared incorporating the active ingredient in the required amount inan appropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan oral solution such as one containing sodium borate, glycerin andpotassium bicarbonate, or dispersed in a dentifrice, or added in atherapeutically-effective amount to a composition that may includewater, binders, abrasives, flavoring agents, foaming agents, andhumectants. Alternatively the compositions may be fashioned into atablet or solution form that may be placed under the tongue or otherwisedissolved in the mouth.

2. Injectable Delivery

In certain circumstances it will be desirable to deliver thepharmaceutical compositions disclosed herein parenterally,intravenously, intramuscularly, or even intraperitoneally as describedin U.S. Pat. No. 5,543,158; U.S. Pat. No. 5,641,515 and U.S. Pat. No.5,399,363 (each specifically incorporated herein by reference in itsentirety). Solutions of the active compounds as free base orpharmacologically acceptable salts may be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions mayalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy syringability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be facilitated by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion (see, e.g., Remington's PharmaceuticalSciences, 15th Edition, pp. 1035-1038 and 1570-1580). Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, and the general safety and purity standards as required byFDA Office of Biologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The compositions disclosed herein may be formulated in a neutral or saltform. Pharmaceutically-acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective. Theformulations are easily administered in a variety of dosage forms suchas injectable solutions, drug-release capsules, and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The phrase “pharmaceutically-acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a human. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared. The preparation can also be emulsified.

3. Nasal Delivery

In certain embodiments, the pharmaceutical compositions may be deliveredby intranasal sprays, inhalation, and/or other aerosol deliveryvehicles. Methods for delivering genes, nucleic acids, and peptidecompositions directly to the lungs via nasal aerosol sprays has beendescribed e.g., in U.S. Pat. No. 5,756,353 and U.S. Pat. No. 5,804,212(each specifically incorporated herein by reference in its entirety).Likewise, the delivery of drugs using intranasal microparticle resins(Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (U.S.Pat. No. 5,725,871, specifically incorporated herein by reference in itsentirety) are also well-known in the pharmaceutical arts. Likewise,transmucosal drug delivery in the form of a polytetrafluoroetheylenesupport matrix is described in U.S. Pat. No. 5,780,045 (specificallyincorporated herein by reference in its entirety).

4. Liposome-, Nanocapsule-, and Microparticle-Medicated Delivery

In certain embodiments, the inventors contemplate the use of liposomes,nanocapsules, microparticles, microspheres, lipid particles, vesicles,and the like, for the introduction of the compositions of the presentinvention into suitable host cells. In particular, the compositions ofthe present invention may be formulated for delivery either encapsulatedin a lipid particle, a liposome, a vesicle, a nanosphere, or ananoparticle or the like.

Such formulations may be preferred for the introduction ofpharmaceutically-acceptable formulations of the nucleic acids orconstructs disclosed herein. The formation and use of liposomes isgenerally known to those of skill in the art (see for example, Couvreuret al., 1977; Couvreur, 1988; Lasic, 1998; which describes the use ofliposomes and nanocapsules in the targeted antibiotic therapy forintracellular bacterial infections and diseases). Recently, liposomeswere developed with improved serum stability and circulation half-times(Gabizon & Papahadjopoulos, 1988; Allen and Choun, 1987; U.S. Pat. No.5,741,516, specifically incorporated herein by reference in itsentirety). Further, various methods of liposome and liposome likepreparations as potential drug carriers have been reviewed (Takakura,1998; Chandran et al., 1997; Margalit, 1995; U.S. Pat. No. 5,567,434;U.S. Pat. No. 5,552,157; U.S. Pat. No. 5,565,213; U.S. Pat. No.5,738,868 and U.S. Pat. No. 5,795,587, each specifically incorporatedherein by reference in its entirety).

Liposomes have been used successfully with a number of cell types thatare normally resistant to transfection by other procedures including Tcell suspensions, primary hepatocyte cultures and PC 12 cells (Renneisenet al., 1990; Muller et al., 1990). In addition, liposomes are free ofthe DNA length constraints that are typical of viral-based deliverysystems. Liposomes have been used effectively to introduce genes, drugs(Heath & Martin, 1986; Heath et al., 1986; Balazsovits et al., 1989;Fresta & Puglisi, 1996), radiotherapeutic agents (Pikul et al., 1987),enzymes (Imaizumi et al., 1990a; Imaizumi et al., 1990b), viruses(Faller & Baltimore, 1984), transcription factors and allostericeffectors (Nicolau & Gersonde, 1979) into a variety of cultured celllines and animals. In addition, several successful clinical trailsexamining the effectiveness of liposome-mediated drug delivery have beencompleted (Lopez-Berestein et al., 1985a; 1985b; Coune, 1988; Sculier etal., 1988). Furthermore, several studies suggest that the use ofliposomes is not associated with autoimmune responses, toxicity orgonadal localization after systemic delivery (Mori & Fukatsu, 1992).

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 Å, containing an aqueous solution in the core.

Liposomes bear resemblance to cellular membranes and are contemplatedfor use in connection with the present invention as carriers for thepeptide compositions. They are widely suitable as both water- andlipid-soluble substances can be entrapped, i.e. in the aqueous spacesand within the bilayer itself, respectively. It is possible that thedrug-bearing liposomes may even be employed for site-specific deliveryof active agents by selectively modifying the liposomal formulation.

In addition to the teachings of Couvreur et al. (1977; 1988), thefollowing information may be utilized in generating liposomalformulations. Phospholipids can form a variety of structures other thanliposomes when dispersed in water, depending on the molar ratio of lipidto water. At low ratios the liposome is the preferred structure. Thephysical characteristics of liposomes depend on pH, ionic strength andthe presence of divalent cations. Liposomes can show low permeability toionic and polar substances, but at elevated temperatures undergo a phasetransition which markedly alters their permeability. The phasetransition involves a change from a closely packed, ordered structure,known as the gel state, to a loosely packed, less-ordered structure,known as the fluid state. This occurs at a characteristicphase-transition temperature and results in an increase in permeabilityto ions, sugars and drugs.

In addition to temperature, exposure to proteins can alter thepermeability of liposomes. Certain soluble proteins, such as cytochromec, bind, deform and penetrate the bilayer, thereby causing changes inpermeability. Cholesterol inhibits this penetration of proteins,apparently by packing the phospholipids more tightly. It is contemplatedthat the most useful liposome formations for antibiotic and inhibitordelivery will contain cholesterol.

The ability to trap solutes varies between different types of liposomes.For example, MLVs are moderately efficient at trapping solutes, but SUVsare extremely inefficient. SUVs offer the advantage of homogeneity andreproducibility in size distribution, however, and a compromise betweensize and trapping efficiency is offered by large unilamellar vesicles(LUVs). These are prepared by ether evaporation and are three to fourtimes more efficient at solute entrapment than MLVs.

In addition to liposome characteristics, an important determinant inentrapping compounds is the physicochemical properties of the compounditself. Polar compounds are trapped in the aqueous spaces and nonpolarcompounds bind to the lipid bilayer of the vesicle. Polar compounds arereleased through permeation or when the bilayer is broken, but nonpolarcompounds remain affiliated with the bilayer unless it is disrupted bytemperature or exposure to lipoproteins. Both types show maximum effluxrates at the phase transition temperature.

Liposomes interact with cells via four different mechanisms: endocytosisby phagocytic cells of the reticuloendothelial system such asmacrophages and neutrophils; adsorption to the cell surface, either bynonspecific weak hydrophobic or electrostatic forces, or by specificinteractions with cell-surface components; fusion with the plasma cellmembrane by insertion of the lipid bilayer of the liposome into theplasma membrane, with simultaneous release of liposomal contents intothe cytoplasm; and by transfer of liposomal lipids to cellular orsubcellular membranes, or vice versa, without any association of theliposome contents. It often is difficult to determine which mechanism isoperative and more than one may operate at the same time.

The fate and disposition of intravenously injected liposomes depend ontheir physical properties, such as size, fluidity, and surface charge.They may persist in tissues for h or days, depending on theircomposition, and half lives in the blood range from min to several h.Larger liposomes, such as MLVs and LUVs, are taken up rapidly byphagocytic cells of the reticuloendothelial system, but physiology ofthe circulatory system restrains the exit of such large species at mostsites. They can exit only in places where large openings or pores existin the capillary endothelium, such as the sinusoids of the liver orspleen. Thus, these organs are the predominate site of uptake. On theother hand, SUVs show a broader tissue distribution but still aresequestered highly in the liver and spleen. In general, this in vivobehavior limits the potential targeting of liposomes to only thoseorgans and tissues accessible to their large size. These include theblood, liver, spleen, bone marrow, and lymphoid organs.

Targeting is generally not a limitation in terms of the presentinvention. However, should specific targeting be desired, methods areavailable for this to be accomplished. Antibodies may be used to bind tothe liposome surface and to direct the antibody and its drug contents tospecific antigenic receptors located on a particular cell-type surface.Carbohydrate determinants (glycoprotein or glycolipid cell-surfacecomponents that play a role in cell-cell recognition, interaction andadhesion) may also be used as recognition sites as they have potentialin directing liposomes to particular cell types. Mostly, it iscontemplated that intravenous injection of liposomal preparations wouldbe used, but other routes of administration are also conceivable.

Alternatively, the invention provides for pharmaceutically-acceptablenanocapsule formulations of the compositions of the present invention.Nanocapsules can generally entrap compounds in a stable and reproducibleway (Henry-Michelland et al., 1987; Quintanar-Guerrero et al., 1998;Douglas et al., 1987). To avoid side effects due to intracellularpolymeric overloading, such ultrafine particles (sized around 0.1 μm)should be designed using polymers able to be degraded in vivo.Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet theserequirements are contemplated for use in the present invention. Suchparticles may be are easily made, as described (Couvreur et al., 1980;1988; zur Muhlen et al., 1998; Zambaux et al. 1998; Pinto-Alphandry etal., 1995 and U.S. Pat. No. 5,145,684, specifically incorporated hereinby reference in its entirety).

Vaccines

In certain preferred embodiments of the present invention, vaccines areprovided. The vaccines will generally comprise one or morepharmaceutical compositions, such as those discussed above, incombination with an immunostimulant. An immunostimulant may be anysubstance that enhances or potentiates an immune response (antibodyand/or cell-mediated) to an exogenous antigen. Examples ofimmunostimulants include adjuvants, biodegradable microspheres (e.g.,polylactic galactide) and liposomes (into which the compound isincorporated; see, e.g., Fullerton, U.S. Pat. No. 4,235,877). Vaccinepreparation is generally described in, for example, Powell & Newman,eds., Vaccine Design (the subunit and adjuvant approach) (1995).Pharmaceutical compositions and vaccines within the scope of the presentinvention may also contain other compounds, which may be biologicallyactive or inactive. For example, one or more immunogenic portions ofother tumor antigens may be present, either incorporated into a fusionpolypeptide or as a separate compound, within the composition orvaccine.

Illustrative vaccines may contain DNA encoding one or more of thepolypeptides as described above, such that the polypeptide is generatedin situ. As noted above, the DNA may be present within any of a varietyof delivery systems known to those of ordinary skill in the art,including nucleic acid expression systems, bacteria and viral expressionsystems. Numerous gene delivery techniques are well known in the art,such as those described by Rolland, Crit. Rev. Therap. Drug CarrierSystems 15:143-198 (1998), and references cited therein. Appropriatenucleic acid expression systems contain the necessary DNA sequences forexpression in the patient (such as a suitable promoter and terminatingsignal). Bacterial delivery systems involve the administration of abacterium (such as Bacillus-Calmette-Guerrin) that expresses animmunogenic portion of the polypeptide on its cell surface or secretessuch an epitope. In a preferred embodiment, the DNA may be introducedusing a viral expression system (e.g., vaccinia or other pox virus,retrovirus, or adenovirus), which may involve the use of anon-pathogenic (defective), replication competent virus. Suitablesystems are disclosed, for example, in Fisher-Hoch et al., Proc. Natl.Acad. Sci. USA 86:317-321 (1989); Flexner et al., Ann. N.Y. Acad. Sci.569:86-103 (1989); Flexner et al., Vaccine 8:17-21 (1990); U.S. Pat.Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No.4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner,Biotechniques 6:616-627 (1988); Rosenfeld et al., Science 252:431-434(1991); Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219 (1994);Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90:11498-11502 (1993);Guzman et al., Circulation 88:2838-2848 (1993); and Guzman et al., Cir.Res. 73:1202-1207 (1993). Techniques for incorporating DNA into suchexpression systems are well known to those of ordinary skill in the art.The DNA may also be “naked,” as described, for example, in Ulmer et al.,Science 259:1745-1749 (1993) and reviewed by Cohen, Science259:1691-1692 (1993). The uptake of naked DNA may be increased bycoating the DNA onto biodegradable beads, which are efficientlytransported into the cells. It will be apparent that a vaccine maycomprise both a polynucleotide and a polypeptide component. Suchvaccines may provide for an enhanced immune response.

It will be apparent that a vaccine may contain pharmaceuticallyacceptable salts of the polynucleotides and polypeptides providedherein. Such salts may be prepared from pharmaceutically acceptablenon-toxic bases, including organic bases (e.g., salts of primary,secondary and tertiary amines and basic amino acids) and inorganic bases(e.g., sodium, potassium, lithium, ammonium, calcium and magnesiumsalts).

While any suitable carrier known to those of ordinary skill in the artmay be employed in the vaccine compositions of this invention, the typeof carrier will vary depending on the mode of administration.Compositions of the present invention may be formulated for anyappropriate manner of administration, including for example, topical,oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous orintramuscular administration. For parenteral administration, such assubcutaneous injection, the carrier preferably comprises water, saline,alcohol, a fat, a wax or a buffer. For oral administration, any of theabove carriers or a solid carrier, such as mannitol, lactose, starch,magnesium stearate, sodium saccharine, talcum, cellulose, glucose,sucrose, and magnesium carbonate, may be employed. Biodegradablemicrospheres (e.g., polylactate polyglycolate) may also be employed ascarriers for the pharmaceutical compositions of this invention. Suitablebiodegradable microspheres are disclosed, for example, in U.S. Pat. Nos.4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763;5,814,344 and 5,942,252. One may also employ a carrier comprising theparticulate-protein complexes described in U.S. Pat. No. 5,928,647,which are capable of inducing a class I-restricted cytotoxic Tlymphocyte responses in a host.

Such compositions may also comprise buffers (e.g., neutral bufferedsaline or phosphate buffered saline), carbohydrates (e.g., glucose,mannose, sucrose or dextrans), mannitol, proteins, polypeptides or aminoacids such as glycine, antioxidants, bacteriostats, chelating agentssuch as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide),solutes that render the formulation isotonic, hypotonic or weaklyhypertonic with the blood of a recipient, suspending agents, thickeningagents and/or preservatives. Alternatively, compositions of the presentinvention may be formulated as a lyophilizate. Compounds may also beencapsulated within liposomes using well known technology.

Any of a variety of immunostimulants may be employed in the vaccines ofthis invention. For example, an adjuvant may be included. Most adjuvantscontain a substance designed to protect the antigen from rapidcatabolism, such as aluminum hydroxide or mineral oil, and a stimulatorof immune responses, such as lipid A, Bortadella pertussis orMycobacterium species or Mycobacterium derived proteins. For example,delipidated, deglycolipidated M. vaccae (“pVac”) can be used. In anotherembodiment, BCG is used as an adjuvant. In addition, the vaccine can beadministered to a subject previously exposed to BCG. Suitable adjuvantsare commercially available as, for example, Freund's Incomplete Adjuvantand Complete Adjuvant (Difco Laboratories, Detroit, Mich.); MerckAdjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 andderivatives thereof (SmithKline Beecham, Philadelphia, Pa.); CWS, TDM,Leif, aluminum salts such as aluminum hydroxide gel (alum) or aluminumphosphate; salts of calcium, iron or zinc; an insoluble suspension ofacylated tyrosine; acylated sugars; cationically or anionicallyderivatized polysaccharides; polyphosphazenes; biodegradablemicrospheres; monophosphoryl lipid A and quil A. Cytokines, such asGM-CSF or interleukin-2, -7, or -12, may also be used as adjuvants.

Within the vaccines provided herein, the adjuvant composition ispreferably designed to induce an immune response predominantly of theTh1 type. High levels of Th1-type cytokines (e.g., IFN-γ, TNFα, IL-2 andIL-12) tend to favor the induction of cell mediated immune responses toan administered antigen. In contrast, high levels of Th2-type cytokines(e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction ofhumoral immune responses. Following application of a vaccine as providedherein, a patient will support an immune response that includes Th1- andTh2-type responses. Within a preferred embodiment, in which a responseis predominantly Th1-type, the level of Th1-type cytokines will increaseto a greater extent than the level of Th2-type cytokines. The levels ofthese cytokines may be readily assessed using standard assays. For areview of the families of cytokines, see Mosmann & Coffman, Ann. Rev.Immunol. 7:145-173 (1989).

Preferred adjuvants for use in eliciting a predominantly Th1-typeresponse include, for example, a combination of monophosphoryl lipid A,preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL), togetherwith an aluminum salt. MPL adjuvants are available from CorixaCorporation (Seattle, Wash.; see U.S. Pat. Nos. 4,436,727; 4,877,611;4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which theCpG dinucleotide is unmethylated) also induce a predominantly Th1response. Such oligonucleotides are well known and are described, forexample, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and5,856,462. Immunostimulatory DNA sequences are also described, forexample, by Sato et al., Science 273:352 (1996). Another preferredadjuvant comprises a saponin, such as Quil A, or derivatives thereof,including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham,Mass.); Escin; Digitonin; or Gypsophila or Chenopodium quinoa saponins.Other preferred formulations include more than one saponin in theadjuvant combinations of the present invention, for example combinationsof at least two of the following group comprising QS21, QS7, Quil A,β-escin, or digitonin.

Alternatively the saponin formulations may be combined with vaccinevehicles composed of chitosan or other polycationic polymers,polylactide and polylactide-co-glycolide particles, poly-N-acetylglucosamine-based polymer matrix, particles composed of polysaccharidesor chemically modified polysaccharides, liposomes and lipid-basedparticles, particles composed of glycerol monoesters, etc. The saponinsmay also be formulated in the presence of cholesterol to formparticulate structures such as liposomes or ISCOMs. Furthermore, thesaponins may be formulated together with a polyoxyethylene ether orester, in either a non-particulate solution or suspension, or in aparticulate structure such as a paucilamelar liposome or ISCOM. Thesaponins may also be formulated with excipients such as Carbopol® toincrease viscosity, or may be formulated in a dry powder form with apowder excipient such as lactose.

In one preferred embodiment, the adjuvant system includes thecombination of a monophosphoryl lipid A and a saponin derivative, suchas the combination of QS21 and 3D-MPL® adjuvant, as described in WO94/00153, or a less reactogenic composition where the QS21 is quenchedwith cholesterol, as described in WO 96/33739. Other preferredformulations comprise an oil-in-water emulsion and tocopherol. Anotherparticularly preferred adjuvant formulation employing QS21, 3D-MPL®adjuvant and tocopherol in an oil-in-water emulsion is described in WO95/17210.

Another enhanced adjuvant system involves the combination of aCpG-containing oligonucleotide and a saponin derivative particularly thecombination of CpG and QS21 as disclosed in WO 00/09159. Preferably theformulation additionally comprises an oil in water emulsion andtocopherol.

Other preferred adjuvants include Montanide ISA 720 (Seppic, France),SAF (Chiron, Calif., United States), ISCOMS (CSL), MF-59 (Chiron), theSBAS series of adjuvants (e.g., SBAS-2, AS2′, AS2,″ SBAS4, or SBAS6,available from SmithKline Beecham, Rixensart, Belgium), Detox (Corixa,Hamilton, Mont.), RC-529 (Corixa, Hamilton, Mont.) and other aminoalkylglucosaminide 4-phosphates (AGPs), such as those described in pendingU.S. patent application Ser. Nos. 08/853,826 and 09/074,720, thedisclosures of which are incorporated herein by reference in theirentireties, and polyoxyethylene ether adjuvants such as those describedin WO 99/52549A1.

Other preferred adjuvants include adjuvant molecules of the generalformula (I): HO(CH₂CH₂O)_(n)-A-R,

wherein, n is 1-50, A is a bond or —C(O)—, R is C₁₋₅₀ alkyl or PhenylC₁₋₅₀ alkyl.

One embodiment of the present invention consists of a vaccineformulation comprising a polyoxyethylene ether of general formula (I),wherein n is between 1 and 50, preferably 4-24, most preferably 9; the Rcomponent is C₁₋₅₀, preferably C₄-C₂₀ alkyl and most preferably C₁₂alkyl, and A is a bond. The concentration of the polyoxyethylene ethersshould be in the range 0.1-20%, preferably from 0.1-10%, and mostpreferably in the range 0.1-1%. Preferred polyoxyethylene ethers areselected from the following group: polyoxyethylene-9-lauryl ether,polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether,polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, andpolyoxyethylene-23-lauryl ether. Polyoxyethylene ethers such aspolyoxyethylene lauryl ether are described in the Merck index (12^(th)edition: entry 7717). These adjuvant molecules are described in WO99/52549.

The polyoxyethylene ether according to the general formula (I) abovemay, if desired, be combined with another adjuvant. For example, apreferred adjuvant combination is preferably with CpG as described inthe pending UK patent application GB 9820956.2.

Any vaccine provided herein may be prepared using well known methodsthat result in a combination of antigen, immune response enhancer and asuitable carrier or excipient. The compositions described herein may beadministered as part of a sustained release formulation (i.e., aformulation such as a capsule, sponge or gel (composed ofpolysaccharides, for example) that effects a slow release of compoundfollowing administration). Such formulations may generally be preparedusing well known technology (see, e.g., Coombes et al., Vaccine14:1429-1438 (1996)) and administered by, for example, oral, rectal orsubcutaneous implantation, or by implantation at the desired targetsite. Sustained-release formulations may contain a polypeptide,polynucleotide or antibody dispersed in a carrier matrix and/orcontained within a reservoir surrounded by a rate controlling membrane.

Carriers for use within such formulations are biocompatible, and mayalso be biodegradable; preferably the formulation provides a relativelyconstant level of active component release. Such carriers includemicroparticles of poly(lactide-co-glycolide), polyacrylate, latex,starch, cellulose, dextran and the like. Other delayed-release carriersinclude supramolecular biovectors, which comprise a non-liquidhydrophilic core (e.g., a cross-linked polysaccharide oroligosaccharide) and, optionally, an external layer comprising anamphiphilic compound, such as a phospholipid (see, e.g., U.S. Pat. No.5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO96/06638). The amount of active compound contained within a sustainedrelease formulation depends upon the site of implantation, the rate andexpected duration of release and the nature of the condition to betreated or prevented.

Any of a variety of delivery vehicles may be employed withinpharmaceutical compositions and vaccines to facilitate production of anantigen-specific immune response that targets tumor cells. Deliveryvehicles include antigen presenting cells (APCs), such as dendriticcells, macrophages, B cells, monocytes and other cells that may beengineered to be efficient APCs. Such cells may, but need not, begenetically modified to increase the capacity for presenting theantigen, to improve activation and/or maintenance of the T cellresponse, to have anti-tumor effects per se and/or to be immunologicallycompatible with the receiver (i.e., matched HLA haplotype). APCs maygenerally be isolated from any of a variety of biological fluids andorgans, including tumor and peritumoral tissues, and may be autologous,allogeneic, syngeneic or xenogeneic cells.

Certain preferred embodiments of the present invention use dendriticcells or progenitors thereof as antigen-presenting cells. Dendriticcells are highly potent APCs (Banchereau & Steinman, Nature 392:245-251(1998)) and have been shown to be effective as a physiological adjuvantfor eliciting prophylactic or therapeutic antitumor immunity (seeTimmerman & Levy, Ann. Rev. Med. 50:507-529 (1999)). In general,dendritic cells may be identified based on their typical shape (stellatein situ, with marked cytoplasmic processes (dendrites) visible invitro), their ability to take up, process and present antigens with highefficiency and their ability to activate naïve T cell responses.Dendritic cells may, of course, be engineered to express specificcell-surface receptors or ligands that are not commonly found ondendritic cells in vivo or ex vivo, and such modified dendritic cellsare contemplated by the present invention. As an alternative todendritic cells, secreted vesicles antigen-loaded dendritic cells(called exosomes) may be used within a vaccine (see Zitvogel et al.,Nature Med. 4:594-600 (1998)).

Dendritic cells and progenitors may be obtained from peripheral blood,bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltratingcells, lymph nodes, spleen, skin, umbilical cord blood or any othersuitable tissue or fluid. For example, dendritic cells may bedifferentiated ex vivo by adding a combination of cytokines such asGM-CSF, IL-4, IL-13 and/or TNFα to cultures of monocytes harvested fromperipheral blood. Alternatively, CD34 positive cells harvested fromperipheral blood, umbilical cord blood or bone marrow may bedifferentiated into dendritic cells by adding to the culture mediumcombinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/orother compound(s) that induce differentiation, maturation andproliferation of dendritic cells.

Dendritic cells are conveniently categorized as “immature” and “mature”cells, which allows a simple way to discriminate between two wellcharacterized phenotypes. However, this nomenclature should not beconstrued to exclude all possible intermediate stages ofdifferentiation. Immature dendritic cells are characterized as APC witha high capacity for antigen uptake and processing, which correlates withthe high expression of Fcγ receptor and mannose receptor. The maturephenotype is typically characterized by a lower expression of thesemarkers, but a high expression of cell surface molecules responsible forT cell activation such as class I and class II MHC, adhesion molecules(e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80,CD86 and 4-1BB).

APCs may generally be transfected with a polynucleotide encoding aprotein (or portion or other variant thereof) such that the polypeptide,or an immunogenic portion thereof, is expressed on the cell surface.Such transfection may take place ex vivo, and a composition or vaccinecomprising such transfected cells may then be used for therapeuticpurposes, as described herein. Alternatively, a gene delivery vehiclethat targets a dendritic or other antigen presenting cell may beadministered to a patient, resulting in transfection that occurs invivo. In vivo and ex vivo transfection of dendritic cells, for example,may generally be performed using any methods known in the art, such asthose described in WO 97/24447, or the gene gun approach described byMahvi et al., Immunology and Cell Biology 75:456-460 (1997). Antigenloading of dendritic cells may be achieved by incubating dendritic cellsor progenitor cells with the polypeptide, DNA (naked or within a plasmidvector) or RNA; or with antigen-expressing recombinant bacterium orviruses (e.g., vaccinia, fowlpox, adenovirus or lentivirus vectors).Prior to loading, the polypeptide may be covalently conjugated to animmunological partner that provides T cell help (e.g., a carriermolecule). Alternatively, a dendritic cell may be pulsed with anon-conjugated immunological partner, separately or in the presence ofthe polypeptide.

Vaccines and pharmaceutical compositions may be presented in unit-doseor multi-dose containers, such as sealed ampoules or vials. Suchcontainers are preferably hermetically sealed to preserve sterility ofthe formulation until use. In general, formulations may be stored assuspensions, solutions or emulsions in oily or aqueous vehicles.Alternatively, a vaccine or pharmaceutical composition may be stored ina freeze-dried condition requiring only the addition of a sterile liquidcarrier immediately prior to use.

Diagnostic Kits

The present invention further provides kits for use within any of theabove diagnostic methods. Such kits typically comprise two or morecomponents necessary for performing a diagnostic assay. Components maybe compounds, reagents, containers and/or equipment. For example, onecontainer within a kit may contain a monoclonal antibody or fragmentthereof that specifically binds to a protein. Such antibodies orfragments may be provided attached to a support material, as describedabove. One or more additional containers may enclose elements, such asreagents or buffers, to be used in the assay. Such kits may also, oralternatively, contain a detection reagent as described above thatcontains a reporter group suitable for direct or indirect detection ofantibody binding.

Alternatively, a kit may be designed to detect the level of mRNAencoding a protein in a biological sample. Such kits generally compriseat least one oligonucleotide probe or primer, as described above, thathybridizes to a polynucleotide encoding a protein. Such anoligonucleotide may be used, for example, within a PCR or hybridizationassay. Additional components that may be present within such kitsinclude a second oligonucleotide and/or a diagnostic reagent orcontainer to facilitate the detection of a polynucleotide encoding aprotein of the invention.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of noncritical parameters that could be changed or modified toyield essentially similar results.

Example 1 Guinea Pig Vaccination with MTB72F Fusion Protein andCompositions with Individual Antigens

Guinea pigs were immunized with adjuvant alone (SBAS1, SBAS2, or ASAS7plus A1(OH)₃), MTB72F fusion protein in adjuvant, or TbH9 plus Ra35antigen composition.

Methods: Groups:  1) SBAS1  2) SBAS2  3) SBAS7 + Al(OH)3  4) TbH9 +Ra35 + SBAS1  5) TbH9 + Ra35 + SBAS2  6) TbH9 + Ra35 + SBAS7(Al(OH)3) 7) MTB72F in SBAS1  8) MTB72F in SBAS2  9) MTB72F in SBAS7 + Al(OH)310) PBS 11) BCG

Dosage: 4 μg each of TbH9 and Ra35

-   -   8 μg MTB72F

Protocol: 1st immunization, 2nd immunization approximately 3 weekslater, 3rd immunization approximately two and a half weeks later.

Pre-challenge: DTH (delayed type hypersensitivity, used to determineantigenicity; 10 μg antigen)

Challenge: Aerosol with ˜30 cfu Erdman strain

Post challenge monitoring: Weight loss

-   -   Death (˜6 months post challenge)

Results:

1. DTH

Positive reaction to the immunizing antigens. Reactions to individualantigens or the fusion protein were comparable. Skin test reactivity toPPD was only seen with the BCG immunized groups

2. Protection: Guinea pigs vaccinated with MTB72F fusion proteinafforded protection compared to those immunized with a mixture ofantigens (see FIG. 1).

Example 2 Mouse Vaccination with MTB72F Fusion Protein and Compositionswith Individual Antigens

As described above, mice were immunized with adjuvant alone (SBAS2,SBAS2′, SBAS2″, or SBAS6), MTB72F fusion protein in adjuvant, MTB72FDNA, MTB59F fusion protein in adjuvant, or TbH9, Ra35 and Ra12 antigencomposition.

Methods: Groups:  1) MTB72F + SBAS2  2) MTB72F + SBAS2′  3) MTB72F +SBAS2″  4) MTB72F + SBAS6  5) Ra12 + TbH9 + Ra35 in SBAS2  6) MTB59F inSBAS2  7) SBAS2  8) MTB72F + delipidated, deglycolipidated M. vaccae  9)MTB72F DNA 10) MTB72F + IFA 11) MTB72F + BCG 12) delipidated,deglycolipidated M. vaccae 13) BCG 14) Saline 15) MTB72F + SBAS2 (inhouse formulation)

8 animals per group

Immunization schedule: First immunization, second immunizationapproximately 3 weeks later; third immunization approximately threeweeks later.

Aerosol challenge approximately three months after first does

Spleen or lung cells were isolated and cultured; count CFU of culturesapproximately three weeks after plating.

Dose: 8 μg MTB72F, 6.56 μg MTB59F, or 1.52, 4.3, and 2.24 μg,respectively, of Ra12, TbH9, and Ra35, mixed.

Results:

Of the AS adjuvants, AS2″+MTB72F gave the best protection in both thespleen and lung in this set of experiments (see FIGS. 2A and 2B). MTB72Fgave ˜1 log better protection than MTB59F in both spleen and lung inthis set of experiments, indicating that Ra12 provides additionalbenefit. Mixture of 12/H9/35+AS2 gave a better protection than MTB72F inthis experiment. MTB72F DNA gave the best protection in this experiment,particularly in the spleen (>2 log). The protection was comparable inthe lung to that seen with MTB72F protein+AS2″, in this experiment.

Example 3 Guinea Pig Vaccination with MTB72F Fusion Protein andCompositions with Individual Antigens

As described above, guinea pigs were immunized with adjuvant alone(SBAS2, SBAS2′, SBAS2″, or SBAS6), MTB72F fusion protein in adjuvant,MTB72F DNA, MTB59F fusion protein in adjuvant, or TbH9, Ra35 and Ra12antigen composition.

Methods: Groups:  1) MTB72F + SBAS2  2) MTB72F + SBAS2′  3) MTB72F +SBAS2″  4) MTB72F + SBAS6  5) Ra12 + TbH9 + Ra35 in SBAS2  6) MTB59F inSBAS2  7) SBAS2  8) MTB72F + pvac  9) MTB72F DNA 10) MTB72F + IFA 11)MTB72F + BCG 12) BCG 13) Saline 14) delipidated, deglycolipidated M.vaccae

Antigens:

Antigens were formulated on a molar equivalent

5 animals per group

Injection volume per dose is 250 μl (IM) containing

MTB72F 20 μg Ra12, TbH9, Ra35 3.8, 10.8, and 5.6 μg MTB59F 16.4 μg

Schedule:

1st immunization, 2nd immunization approximately three weeks later, 3rdimmunization approximately three weeks later.

Challenge: ˜one and one half months after first immunization.

Results:

˜38 Wks post challenge

Groups Alive State G1. MTB72F + AS2 1/5 [losing weight] G2. MTB72F +AS2′ 2/5 [not gaining weight] G3. MTB72F + AS2″ 3/5 [looking okay, butno weight gain] G4. MTB72F + AS6 2/5 [both these gaining weight] G5.MTBRa12 + H9 + 4/5 [one maybe a bit peaked, Ra35 + AS2 but two gaining]G6. MTB59F + AS2 2/5 [both losing a little] G7. AS2 2/5 [both losing]G8. MTB72F + pVac 1/5 [not looking too good] G9. MTB72F DNA 3/5 [allholding steady] G10. MTB72F + IFA 2/5 [doing okay] G11. MTB72F + BCG 5/5[eating very well] G12 BCG 4/5 [doing fine] G13 Saline all dead G14 pVac2/5 [not gaining weight]

By 50 weeks post challenge, while 80% (4/5) of the guinea pigs immunizedwith BCG+Mtb72F were still alive, only 20% (1/5) of those immunized withBCG alone were alive. At 85 weeks, 4/5 of the guinea pigs immunized withBCG+Mtb72F were still alive and healthy (see FIG. 7).

Example 4 Long Term Protection

As described above, guinea pigs were immunized with adjuvant alone (AS2or AS2″), MTB72F fusion protein in adjuvant, TbH9, Ra35 and Ra12 antigencomposition, or a variety of individual antigens in adjuvant.

METHODS GROUPS ANTIGEN DOSE 1. AS2″ + MTB39 (TbH9) 20ug/250ul (IM) 2.AS2″ + MTB8.4 (DPV) 20ug 3. AS2″ + MTB9.9 (MTI) 20ug 4. AS2″ + MTB41(MTCC#2) 20ug 5. AS2″ + MTB40 (HTCC#1) 20ug 6. AS2″ + MTB9.8 (MSL) 20ug7. AS2″ + MTB72F 20ug 8. AS2″ + Ra12 + TbH9 + Ra35 3.8 μg + 10.8 μg +5.6 μg (molar equivalent) 9. AS2″ + MTB71F + 20 μg + 20 μg + 10 μgMTB72F + HTCC#1 10. AS2″ + Ra12 20 μg 11. BCG 12. AS2″ 13. AS2 + MTB72F14. AS2 + Ra12 + TbH9 + Ra35 15. AS2

Example 5 Monkey Vaccination with MTB72F Fusion Protein and Compositionswith Individual Antigens

As described above, monkeys were immunized with MTB72F fusion protein inSBAS2 adjuvant, or MTB8.4 antigen composition in adjuvant, or a mixtureof MTB72F and MTB8.4.

Methods: Groups 1. Saline 2. BCG 3. MTB8.4/AS2 4. MTB72F/AS2 5.MTB72F/AS2 (one arm) + MTB8.4/AS2 (other arm) 40 μg each antigen

Results:

At 8 weeks post challenge, monkeys immunized with BCG are showing signsof infection

Current data for 16 weeks post challenge reveals the following trend:

Groups immunized with MTB72F (4 and 5) are holding on their weights andhave low ESR values compared to group 3 (MTB8.4 immunization) (Tables 1and 2).

TABLE 1 Prophylactic Vaccine Study in Cynomolgus Monkeys with MTB8.4 andMTB72F formulated in AS2 20 Weeks Post Challenge Net weight Groups IDChange (kg) Chest X-ray (onset) Status AS2 1398K   −24% Pn, bil, prog(wk 8) Alive 4437B   −33% Pn, bil, prog (wk4) Dead 2959G  −8.30% Pn,bil, prog (wk4) Alive 605AE −14.00% Pn, rt, stable (wk 8) Alive BCG3436A −15.00% Neg Alive 3642G Plus 4.5% Pn, rt, prog (wk 8) Alive 1190H   0% Neg Alive 1051I   −30% Pn, rt, prog (wk 8) Dead MTB8.4 3665C  −25% Pn, rt, prog (wk8) Dead 2200F −18.00% Pn, rt, stable (wk8) Alive1654J −33.00% Pn, bil, prog (wk4) Dead 4141C   −33% Pn, bil, prog (wk4)Dead MTB72F 3061C* Died after IT challenge 1228G Plus 3.6% Bron, bil,stable for 3 Alive mo (wk8) 3462E  −2.20% Neg Alive 4254C Plus 1.21 Pn,rt, stable for 3 mo Alive (wk4) MTB8.4 4496A Plus 7% Pn, rt, stable for1 mo Alive (wk 8) MTB72F 4422C −39.00% Pn, bil, prog (wk 4) Dead 4416APlus 11% Pn, rt, stable for 2 mo Alive (wk 12) 2734E Plus 12.5% Suspinfil rt, stable for 3 Alive mo (wk 8)

TABLE 2 Prophylactic Vaccine Study in Cynomolgus Monkeys with MTB8.4 andMTB72F formulated in AS2 Wks Post Challenge ESR Groups ID 4 8 12 16 16wks Chest X-ray AS2 1398K 3 3 10 19 Pn, bil, progrsv 4437B 10 20 3 Died2959G 6 3 3 0 Pn, rt, progrsv 605AE 1 4 7 3 Pn, rt, stable BCG 3436A 0 87 15 Neg 3642G 0 0 0 0 Pn, rt, progrsv 1190H 1 0 2 0 Neg 1051I 0 8 22 7Pn, bil, w/furt progrsn Died MTB8.4 3665C 12 30 19 Died 2200F 1 7 2 0Pn, rt, progrsv 1654J 20 8 21 7 Pn, bil, w/fur progrsn 4141C 13 8 2 15Pn, bil, w/fur progrsn MTB72F 3061C* Died after IT challenge 1228G 0 120 0 Now stable 3462E 0 0 0 0 Neg 4254C 13 0 0 0 Pn, now stable MTB8.4/4496A 5 1 0 5 Pu, rt, w/furt prog MTB72F 4422C 10 3 0 Died 4416A 6 0 1 0Pu, now stable 2734E 0 0 0 0 Susp infil, now stable

Example 6 BCG Priming Experiment in Monkeys

5 animals per group with four groups immunized with BCG and then rested,then immunized as described above and challenged. The following protocolwill be used:

Groups # animals Immunizing Antigen Antigen Dose 1. Nothing 5 AS2 2. BCG5 AS2 3. BCG 5 MTB72F 40ug 4. BCG 4 Ra12 + TbH9 + Ra35 Molar equiv ofantigens in MTB72F dose 5. BCG 4 MTB72F + MTB71F + 40ug MTB72F MTB4040ug MTB72F 20ug MTB40

All antigens in formulated in AS2

Groups 4 and 5 have four animals each. Two of the BCG immunized monkeysdied

Immunizing Antigen proliferation and cytokine Groups # animalsproduction assays Antigens for T cell 1. Nothing 5 AS2 PHA, PPD, MTB72F,MTB71F, HTCC#1, DPV, MTCC#2, Ra12, TbH9, Ra35, MSL, MTI 2. BCG 5 AS2PHA, PPD, MTB72F, MTB71F, HTCC#1, DPV, MTCC#2, Ra12, TbH9, Ra35, MSL,MTI 3. BCG 5 MTB72F PHA, PPD, MTB72F, Ra12, TbH9, Ra35 4. BCG 4 Ra12 +TbH9 + Ra35 PHA, PPD, MTB72F, Ra12, TbH9, Ra35 5. BCG 4 MTB72F +MTB71F + MTB40 PHA, PPD, MTB72F, MTB71F, HTCC#1, DPV, MTCC-2, Ra12,TbH9, Ra35, MSL, MTI

Example 7 Construction of Ra35MutSA and MTB72FMutSA

Expression of Mtb72f typically results in some breakdown products. Inaddition, the expression of the full-length sequences of the mature orfull length form of Ra35 (Mtb32A) in E. coli has been difficult. Theexpressed product was only visible after immunoblotting with apolyclonal rabbit anti-Ra35Ab indicative of low levels of proteinexpression. Even then, multiple specific species (bands) were detectedindicative of auto-catalytic breakdown (degradation) of the recombinantantigen. This was presumed to be due to the expression of Ra35FL in E.coli as a biologically active form.

It has been previously shown that it was possible to express Ra35FL astwo overlapping halves comprising the N-terminal (Ra35N-term, calledRa35) and C-term halves (Ra35C-term called Ra12). To enhance andstabilize the expression of the whole Ra35 molecule, a single pointmutation was introduced at one of the residues within the active-sitetriad (substitution of Ser to Ala; see FIG. 6). This mutagenized form ofMtb32A can now be easily expressed at high levels in a stable form. Inaddition, to stabilize expression of Mtb72F, a single nucleotidesubstitution (T to G, resulting in a Ser to Ala change at position 710of the fusion polypeptide) was incorporated in the sequence of Mtb72F atnucleotide position 2128 (see FIG. 5).

This stabilization is also readily accomplished by mutagenizing any one,any two, or all three of the three residues comprising the active sitetriad in Ra35FL, Ra35, or Mtb72F or other fusion proteins comprisingRa35 (His, Asp, or Ser). Mutagenesis can be performed using anytechnique known to one of skill in the art.

Example 8 Immunization of mice with f Ra35FLMutSA-TbH9 and MTB72FMutSA

Eight mice per group were immunized with the compositions listed, below,which include the adjuvant AS2A. The mice were then challenged withMycobacterium tuberculosis, and survival of the mice was measured.

Group Concentration of protein or DNA 1. Mtb72f protein 1.5 mg/ml 2.Mtb72f DNA 1.2 mg/ml 3. Mtb72f-85b protein 0.6 mg/ml 4. Mtb72f-85b DNA1.1 mg/ml 5. Mtb72f-MTI protein 1.3 mg/ml 6. Mtb72f-MTI DNA 1.1 mg/ml 7.Mtb72fMutSA protein 1.7 mg/ml 8. MTB3AMutSA-TbH9 protein 2.4 mg/ml 9.BCG 10. AS2 11. vector alone 1.5 mg/ml 12. saline

1-88. (canceled)
 89. A polynucleotide comprising a nucleotide sequenceencoding a fusion polypeptide, said fusion polypeptide comprising afirst amino acid sequence having at least 95% sequence identity to SEQID NO:14 and a second amino acid sequence having at least 95% sequenceidentity to SEQ ID NO:8, and wherein the amino acid corresponding toposition 176 in SEQ ID NO:8 is not a serine.
 90. The polynucleotide ofclaim 89, wherein the encoded fusion polypeptide further comprises athird amino acid sequence having at least 95% sequence identity to SEQID NO:10.
 91. The polynucleotide of claim 89 or 90, wherein the encodedfusion has an amino acid corresponding to position 176 in SEQ ID NO:8which is an alanine.
 92. The polynucleotide of claim 89, wherein theencoded fusion polypeptide has at least 95% sequence identity to SEQ IDNO:18 or SEQ ID NO:20.
 93. The polynucleotide of claim 89, wherein theencoded fusion polypeptide comprises the amino acid sequence of SEQ IDNO:14 and the amino acid sequence of SEQ ID NO:8, and wherein the aminoacid corresponding to position 176 in SEQ ID NO:8 is not a serine. 94.The polynucleotide of claim 93, wherein the encoded fusion polypeptidefurther comprises the amino acid sequence of SEQ ID NO:10.
 95. Thepolynucleotide of claim 93 or 94, wherein the encoded fusion has anamino acid corresponding to position 176 in SEQ ID NO:8 which is analanine.
 96. The polynucleotide of claim 93 or 94, wherein the encodedfusion polypeptide further comprises a 6× histidine tag.
 97. Thepolynucleotide of claim 89, wherein the encoded fusion polypeptidecomprises the amino acid sequence of SEQ ID NO:18.
 98. Thepolynucleotide of claim 89, wherein the encoded fusion polypeptidecomprises the amino acid sequence of SEQ ID NO:20 with an alaninereplacing the serine at position
 577. 99. A recombinant expressioncassette comprising the polynucleotide of claim
 89. 100. The expressioncassette of claim 99, which is a viral vector.
 101. The expressioncassette of claim 99, the polynucleotide comprising a nucleotidesequence encoding a fusion polypeptide having at least 95% sequenceidentity to SEQ ID NO: 18 or SEQ ID NO:20.
 102. The expression cassetteof claim 101, which is a viral vector.
 103. A composition comprising thepolynucleotide of claim
 89. 104. The composition of claim 103, thepolynucleotide comprising a nucleotide sequence encoding a fusionpolypeptide having at least 95% sequence identity to SEQ ID NO:18 or SEQID NO:20.
 105. The composition of claim 103, further comprising anadjuvant.
 106. The composition of claim 104, further comprising anadjuvant.
 107. A method for the treatment and/or prevention oftuberculosis comprising administering an effective amount of thepolynucleotide of claim
 89. 108. The method of claim 107, comprisingadministering an effective amount of a polynucleotide comprising anucleotide sequence encoding a fusion polypeptide having at least 95%sequence identity to SEQ ID NO:18 or SEQ ID NO:20.
 109. A method ofrecombinantly making a fusion protein, the method comprising the step ofexpressing the polynucleotide of claim 1 in a host cell.
 110. The methodof claim 109, the method comprising the step of expressing apolynucleotide comprising a nucleotide sequence encoding a fusionpolypeptide having at least 95% sequence identity to SEQ ID NO:18 or SEQID NO:20 in a host cell.